Droplet-based affinity assays

ABSTRACT

A method of detecting a target analyte in a sample, including executing electrowetting-mediated droplet operations and thereby: combining one or more immunoassay reagent droplets comprising magnetically-responsive beads having affinity for the target analyte with one or more sample droplets potentially comprising the target analyte to yield a first combined droplet; beginning with the combined droplet, effecting a droplet-based washing protocol to wash the magnetically-responsive beads to yield a first washed droplet comprising the washed magnetically responsive beads; and combining the droplet comprising the washed magnetically responsive beads with a droplet comprising a reporter molecule having affinity for target analyte captured on the magnetically-responsive beads to yield a second combined droplet; beginning with the second combined droplet, effecting a droplet-based washing protocol to wash the magnetically-responsive beads to yield a second washed droplet comprising the washed magnetically responsive beads; detecting a signal from the second washed droplet which corresponds to the presence, absence and/or quantity of the analyte in the sample.

2 RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/639,663, entitled “Droplet Based Affinity Assays,” filed Dec. 15,2006, which is claims priority to and incorporates by referenceprovisional U.S. Patent Application Nos. 60/744,780, entitled “Apparatusand Methods for Droplet-Based Protein Crystallization,” filed on Apr.13, 2006; 60/745,058, entitled “Filler Fluids for Droplet-BasedMicrofluidics” filed on Apr. 18, 2006; 60/745,049, entitled “Apparatusand Methods for Droplet-Based Protein Crystallization,” filed on Apr.18, 2006; 60/745,039, entitled “Apparatus and Methods for Droplet-BasedBlood Chemistry,” filed on Apr. 18, 2006; 60/745,073, entitled“Apparatus and Methods for Droplet-Based PCR,” filed on Apr. 18, 2006;60/745,059, entitled “Apparatus and Methods for Droplet-BasedImmunoassay,” filed on Apr. 18, 2006; 60/745,914, entitled “Apparatusand method for Manipulating Droplets with a Predetermined Number ofCells” filed on Apr. 28, 2006; 60/745,950, entitled “Apparatus andMethods of Sample Preparation for a Droplet Microactuator,” filed onApr. 28, 2006; 60/746,797 entitled “Portable Analyzer UsingDroplet-Based Microfluidics,” filed on May 9, 2006; 60/746,801, entitled“Apparatus and Methods for Droplet-Based Immuno-PCR,” filed on May 9,2006; 60/745,054, entitled “Droplet-Based Multi-Well Plate, filed onApr. 18, 2006; 60/806,400, entitled “Droplet Microactuator StampingPlatform,” filed on Jun. 30, 2006; 60/806,412, entitled “Systems andMethods for Droplet Microactuator Operations,” filed on Jun. 30, 2006;60/807,104, entitled “Method and Apparatus for Droplet-Based NucleicAcid Amplification,” filed on Jul. 12, 2006.

1 GRANT INFORMATION

This invention was made with government support under CA 114993 awardedby National Institutes of Health. The government has certain rights inthe invention.

3 FIELD OF THE INVENTION

The invention relates to a droplet microactuator and to systems,apparatuses and methods employing the droplet microactuator forexecuting various protocols using discrete droplets. More particularly,the invention relates to droplet-based affinity assays.

4 BACKGROUND OF THE INVENTION

The capability to rapidly conduct biochemical and other assays iscritical in a wide variety of fields. For example, rapid and accuratediagnosis of infectious disease is crucial both for the effectivemanagement of disease in individual subjects and for ameliorating thepublic health problems of multi-drug resistant pathogens and communityacquired infections.

Current PCR-based DNA amplification methods suffer from a number ofdrawbacks including high reagent costs, labor intensity andsusceptibility to cross-contamination. Furthermore, compared to culture,PCR tests are less capable of simultaneously assaying multiple species,virulence factors, and drug resistant markers. They often lacksensitivity and cost-effective quantification of the pathogen. There isa need in the art for improved devices for nucleic acid detection thatwould overcome these limitations while also miniaturizing and automatingthe technique so that these assays could potentially be applied at thepoint-of-sample collection with minimal training.

Nucleic acid sequencing is becoming increasingly common in a variety offields, such as whole genome sequencing, diagnostics, pharmacogenomics,and forensics. However, the sequencing field has been hampered by theexpensive nature of sequencing machines. The development of inexpensive,high-throughput testing systems is critically important to the spread ofgenetic testing and the many advantages that are associated with it.There is thus a need for new technological platforms that allow one toquickly and reliably sequence nucleic acids at a reasonable cost. Theinvention described herein provides an inexpensive, droplet-basedsequencing system.

Immunoassays are widely used for clinical diagnostics and constitutemore than a $3 billion market in the US alone. Immunoassays are amongthe most sensitive and specific methods that are routinely used in aclinical laboratory. Immunoassays make use of the high-affinity andspecificity in binding between an antigen and its homologous antibody todetect and quantify the antigen in a sample matrix. Heterogeneousimmunoassays such as ELISA (Enzyme-Linked Immunosorbent Assay) are amongthe most sensitive and specific clinical analysis methods, and have beenwidely used for identification of a large class of antigens andantibodies. For example, immunoassays are performed, among other things,for identification of cardiac markers, tumor markers, drugs, hormones,and infectious diseases.

Small sample consumption, faster analysis, and complete automation arethree highly desirable features that require continual improvement inany clinical analyzer. Although state-of-the-art laboratory immunoassayanalyzers offer good automation and throughput, they require asignificant amount of sample per test (including dead volumes) andlengthy analysis times. The long assay times and the large size of theseanalyzers make them impractical for use in a point-of-sample collectionsetting.

Also, there is considerable variability in the immunoassay performance,in large part attributed to the techniques being operator dependent,resulting in difficulty comparing results from study to study and evenwithin the same study if more than one laboratory is used. A fullyautomated and integrated analyzer that eliminates the operatordependence and standardizes results for the immune monitoring assayswould considerably improve the interpretation of results from assays.

Though significant advances have been made in the automation ofimmunoassays, these analyzers are prohibitively expensive and are notaffordable in a low-throughput research setting. Lower end systems withautomated plate washers, incubators and integrated optics still requirea skilled technician to perform several key steps in an immunoassay suchas preparing microtiter plates with antibodies and loading samples ontothe plates. This results in human error due to repeated manualintervention and is a major source of inter-assay and intra-assayvariation.

There is also a need for point of sample collection testing in a varietyof fields, such as medicine, environmental, and bioterrorism-relateddetection fields. As an example, point-of-sample collection (POC)testing for bedside blood analysis has improved but remains a keychallenge for modern medical care. Ideally, POC testing would enable theclinicians to diagnose and implement life-saving technologies inreal-time by avoiding the need for large laboratory facilities. Thereremains a need in the art for a lab-on-a-chip that enables simultaneousmonitoring of blood gases, metabolites, electrolytes, enzymes, DNA,proteins, and cells, on low sample volumes at the POC.

Microfluidic control of the fluids is an essential requirement for asuccessful lab-on-a-chip. Microfluidic systems can be broadly groupedinto continuous-flow and discrete-flow based architectures. As the namesuggests, continuous-flow systems rely on continuous flow of liquids inchannels whereas discrete-flow systems utilize droplets of liquid eitherwithin channels or in a channel-less architecture. A common limitationof continuous flow systems is that liquid transport is physicallyconfined to permanently fixed channels. The transport mechanisms usedare usually pressure-driven by external pumps orelectrokinetically-driven by high-voltages. These approaches involvecomplex channeling and require large supporting systems in the form ofexternal valves or power supplies. These restrictions make it difficultto achieve a high degree of functional integration and control inconventional continuous-flow systems, particularly in realizing ahandheld device at the point-of-sample collection. There remains a needin the art for a point of sample collection testing system that makesuse of droplet manipulations and especially a system that can accomplishmultiple tests or multiple types of tests on a single chip.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to droplet-based affinity assays.According to one embodiment, a method of detecting a target analyte in asample is provided, wherein the method comprises: (a) executing dropletoperations to combine affinity-based assay reagents on a dropletmicroactuator with a sample potentially comprising the target analyte togenerate a signal indicative of the presence, absence and/or quantity ofanalyte; and (b) detecting the signal, wherein the signal corresponds tothe presence, absence and/or quantity of the analyte in the sample.

6 DEFINITIONS

As used herein, the following terms have the meanings indicated.

“Activate” with reference to one or more electrodes means effecting achange in the electrical state of the one or more electrodes whichresults in a droplet operation.

“Affinity” means the specific or non-specific intramolecular attractionof one molecule for another molecule or for a substrate, such as theattraction of an antibody for its corresponding antigen or hapten.

“Analyte,” means a target substance for detection which may be presentin a sample. Illustrative examples include antigenic substances,haptens, antibodies, proteins, peptides, amino acids, nucleotides,nucleic acids, drugs, ions, salts, small molecules, cells.

“Antibody” means a polypeptide that has affinity for an epitope orhapten. An antibody can be a polyclonal antibody, a monoclonal antibody,an antibody fragment, and/or an engineered molecule capable of bindingthe corresponding member of a specific binding pair. Antibodies may belabeled or otherwise conjugated to molecules that facilitate direct orindirect detection of and/or quantification of the antibody.

“Bead,” with respect to beads on a droplet microactuator, means any beador particle capable of interacting with a droplet on or in proximitywith a droplet microactuator. Beads may be any of a wide variety ofshapes, such as spherical, generally spherical, egg shaped, disc shaped,cubical and other three dimensional shapes. The bead may, for example,be capable of being transported in a droplet on a droplet microactuator;configured with respect to a droplet microactuator in a manner whichpermits a droplet on the droplet microactuator to be brought intocontact with the bead, on the droplet microactuator and/or off thedroplet microactuator. Beads may be manufactured using a wide variety ofmaterials, including for example, resins, and polymers. The beads may beany suitable size, including for example, microbeads, microparticles,nanobeads and nanoparticles. In some cases, beads are magneticallyresponsive; in other cases beads are not significantly magneticallyresponsive. For magnetically responsive beads, the magneticallyresponsive material may constitute substantially all of a bead or onlyone component of a bead. The remainder of the bead may include, amongother things, polymeric material, coatings, and moieties which permitattachment of an assay reagent. Examples of suitable magneticallyresponsive beads are described in U.S. Patent Publication No.2005-0260686, “Multiplex flow assays preferably with magnetic particlesas solid phase,” published on Nov. 24, 2005, the entire disclosure ofwhich is incorporated herein by reference for its teaching concerningmagnetically responsive materials and beads.

“dNTP” means deoxynucleotidetriphosphate, where the nucleotide is anynucleotide, such as A, T, C, G or U. “ddNTP” meansdideoxynucleotidetriphosphate, where the nucleotide is any nucleotide,such as A, T, C, G or U. It will be appreciated that unless otherwisespecifically indicated, ddNTP can be substituted for dNTP, and viceversa.

“Droplet” means a volume of liquid on a droplet microactuator which isat least partially bounded by filler fluid. For example, a droplet maybe completely surrounded by filler fluid or may be bounded by fillerfluid and one or more surfaces of the droplet microactuator. Dropletsmay take a wide variety of shapes; nonlimiting examples includegenerally disc shaped, slug shaped, truncated sphere, ellipsoid,spherical, partially compressed sphere, hemispherical, ovoid,cylindrical, and various shapes formed during droplet operations, suchas merging or splitting or formed as a result of contact of such shapeswith one or more surfaces of a droplet microactuator.

“Droplet operation” means any manipulation of a droplet on a dropletmicroactuator. A droplet operation may, for example, include: loading adroplet into the droplet microactuator; dispensing one or more dropletsfrom a source droplet; splitting, separating or dividing a droplet intotwo or more droplets; transporting a droplet from one location toanother in any direction; merging or combining two or more droplets intoa single droplet; diluting a droplet; mixing a droplet; agitating adroplet; deforming a droplet; retaining a droplet in position;incubating a droplet; heating a droplet; vaporizing a droplet; cooling adroplet; disposing of a droplet; transporting a droplet out of a dropletmicroactuator; other droplet operations described herein; and/or anycombination of the foregoing. The terms “merge,” “merging,” “combine,”“combining” and the like are used to describe the creation of onedroplet from two or more droplets. It should be understood that whensuch a term is used in reference to two or more droplets, anycombination of droplet operations sufficient to result in thecombination of the two or more droplets into one droplet may be used.For example, “merging droplet A with droplet B,” can be achieved bytransporting droplet A into contact with a stationary droplet B,transporting droplet B into contact with a stationary droplet A, ortransporting droplets A and B into contact with each other. The terms“splitting,” “separating” and “dividing” are not intended to imply anyparticular outcome with respect to size of the resulting droplets (i.e.,the size of the resulting droplets can be the same or different) ornumber of resulting droplets (the number of resulting droplets may be 2,3, 4, 5 or more). The term “mixing” refers to droplet operations whichresult in more homogenous distribution of one or more components withina droplet. Examples of “loading” droplet operations includemicrodialysis loading, pressure assisted loading, robotic loading,passive loading, and pipette loading.

“Electronically coupled” is used herein to indicate an electrical ordata relationship between two or more components or elements. As such,the fact that a first component is said to be electronically coupled toa second component is not intended to exclude the possibility thatadditional components may be present between, and/or operativelyassociated or engaged with, the first and second components. Further,electrically coupled components may in some embodiments include wirelessintervening components.

“Highlight” used with reference to a user interface or the like, such asa droplet microactuator map as described herein, means that a componentof the user interface or map may be visually differentiated, e.g., by achange in color, brightness, shading, shape, or byappearance/disappearance of a symbol, icon, or other visual identifier.For example, mousing over or selecting a representation of an electrodeon the user interface or may cause the electrode representation tochange color. Sounds may also accompany highlighted items to furtherfacilitate user interaction with the system.

“Input device” is used broadly to include all possible types of devicesand ways to input information into a computer system or onto a network.Examples include stylus-based devices, pen-based devices, keyboarddevices, keypad devices, touchpad devices, touch screen devices,joystick devices, trackball devices, mouse devices, bar-code readerdevices, magnetic strip reader devices, infrared devices, speechrecognition technologies.

“Magnetically responsive” means responsive to a magnetic field. Examplesof magnetically responsive materials include paramagnetic materials,ferromagnetic materials, ferrimagnetic materials, and metamagneticmaterials. Examples of suitable paramagnetic materials include iron,nickel, and cobalt, as well as metal oxides such as Fe₃O₄, BaFe₁₂O₁₉,CoO, NiO, Mn₂O₃, Cr₂O₃, and CoMnP.

“Output device” is used broadly to include all possible types of devicesand ways to output information or data from a computer system to a useror to another system. Examples include visual displays, LEDs, printers,speakers, modems and wireless transceivers.

“Protocol” means a series of steps that includes, but is not limited to,droplet operations on one or more droplet microactuators.

“Select” with reference to a user interactive element, such as icon,field, or virtual button, displayed on a user interface means to provideinput which results in the execution of instructions associated with theobject. Thus, for example, selection of a representation of an electrodedisplayed on a droplet microactuator map by pointing and clicking on theelectrode representation may result in execution of instructionsnecessary for activating the actual electrode and/or instructionsnecessary for adding a line of code to a set of instructions whichinstructs activation of the actual electrode. Selection may be achievedusing any of a variety of input devices or combination of input devices,such as mouse, joystick, and/or keyboard.

“Surface” with reference to immobilization of a molecule, such as anantibody or in analyte, on the surface, means any surface on which themolecule can be immobilized while retaining the capability to interactwith droplets on a droplet microactuator. For example, the surface maybe a surface on the droplet microactuator, such as a surface on the topplate or bottom plate of the droplet microactuator; a surface extendingfrom the top plate or bottom plate of the droplet microactuator; asurface on a physical object positioned on the droplet microactuator ina manner which permits it to interact with droplets on the dropletmicroactuator; and/or a bead positioned on the droplet microactuator,e.g., in a droplet and/or in a droplet microactuator but exterior to thedroplet.

“Washing” with respect to washing a surface means reducing the amount ofone or more substances in contact with the surface or exposed to thesurface from a droplet in contact with the surface. The reduction in theamount of the substance may be partial, substantially complete, or evencomplete. The substance may be any of a wide variety of substances;examples include target substances for further analysis, and unwantedsubstances, such as components of a sample, contaminants, and/or excessreagent. The surface may, for example, be a surface of a dropletmicroactuator or a surface of a bead on a droplet microactuator. In someembodiments, a washing operation begins with a starting droplet incontact with a surface, where the droplet includes an initial totalamount of a substance. The washing operation may proceed using a varietyof droplet operations. The washing operation may yield a droplet incontact with the surface, where the droplet has a total amount of thesubstance which is less than the initial amount of the substance. Inanother embodiment, the droplet operation may yield the surface in theabsence of a droplet, where the total amount of the substance in contactwith the surface or otherwise exposed to the surface is less than theinitial amount of the substance in contact with the surface or exposedto the surface in the starting droplet. Other embodiments are describedelsewhere herein, and still others will be immediately apparent in viewof the present disclosure.

When a given component such as a layer, region or substrate is referredto herein as being disposed or formed “on” another component, that givencomponent can be directly on the other component or, alternatively,intervening components (for example, one or more coatings, layers,interlayers, electrodes or contacts) can also be present. It will befurther understood that the terms “disposed on” and “formed on” are usedinterchangeably to describe how a given component is positioned orsituated in relation to another component. Hence, the terms “disposedon” and “formed on” are not intended to introduce any limitationsrelating to particular methods of material transport, deposition, orfabrication.

When a liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a dropletmicroactuator, it should be understood that the droplet is arranged onthe droplet microactuator in a manner which facilitates using thedroplet microactuator to conduct droplet operations on the droplet, thedroplet is arranged on the droplet microactuator in a manner whichfacilitates sensing of a property of or a signal from the droplet,and/or the droplet has been subjected to a droplet operation on thedroplet microactuator.

7 BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top plan view of a droplet microactuator for use inamplification protocols in accordance with an embodiment of the presentinvention;

FIGS. 2A and 2B are top plan views of a droplet microactuator with asingle integrated heater and a plurality of integrated heaters,respectively, in accordance with various embodiments of the presentinvention;

FIG. 3 is a top plan view of a droplet microactuator for use in nucleicacid sequence analysis in accordance with an embodiment of the presentinvention;

FIGS. 4 and 5 are illustrations showing reaction steps and dropletoperations of an illustrative embodiment in accordance with the presentinvention;

FIG. 6 is a perspective view of a droplet microactuator for use inconducting immunoassays in accordance with an embodiment of the presentinvention;

FIG. 7 is an illustration showing steps for conducting a droplet-basedsandwich affinity-based assay performed on a droplet microactuator inaccordance with an embodiment of the present invention;

FIG. 8 is an illustration showing steps for conducting a competitiveaffinity-based assay performed on a droplet microactuator in accordancewith an embodiment of the present invention;

FIG. 9 is a perspective view of a biological fluid analyzer inaccordance with an embodiment of the present invention;

FIG. 10 is a side profile view of a droplet microactuator loadingstructure in accordance with an embodiment of the present invention;

FIGS. 11-13 are illustrations showing steps for immobilizing and freeingmagnetically responsive beads using a magnetic field in accordance withvarious embodiments of the present invention;

FIG. 14 is an illustration showing steps for immobilizing and freeingbeads using a physical obstacle in accordance with an embodiment of thepresent invention;

FIG. 15 is an illustration showing steps for washing a dropletmicroactuator surface in accordance with an embodiment of the presentinvention;

FIG. 16A is a side profile view and FIG. 16B is a top plan view of adroplet microactuator for transporting droplets in accordance with anembodiment of the present invention;

FIG. 17 is a perspective view of a biological fluid analyzer inaccordance with an embodiment of the present invention;

FIG. 18 is an illustration of droplet microactuator systems inaccordance with an embodiment of the present invention;

FIGS. 19A and 19B are illustrations of a portable hand-held analyzer inaccordance with an embodiment of the present invention;

FIG. 20 is an illustration of a user interface of a droplet controlsystem in accordance with an embodiment of the present invention; and

FIGS. 21A-21D is a side profile view illustrating various dropletmicroactuator sensor element configurations in accordance with variousembodiments of the present invention.

8 DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods, devices and systems for executing one ormore droplet-based biochemical assays. For example, the inventionprovides methods, devices and systems for amplifying nucleic acids,analyzing the sequences of nucleic acids, conducting affinity-basedassays, and/or analyzing components of bodily fluids.

In certain embodiments, a protocol of the system may involve one or moreof the following steps in any order which achieves the detection end ofthe invention: extracting sample from a subject; processing the samplefor loading onto a droplet microactuator; loading the sample onto thedroplet microactuator; dispensing one or more sample droplets of thesample for transport on the droplet microactuator; loading one or morereagents onto the droplet microactuator; dispensing one or more reagentdroplets for transport on the droplet microactuator; transporting one ormore reagent droplets and/or one or more sample droplets so as to bringthe one or more reagent droplets into contact with the one or moresample droplets thereby effecting interaction of the reagent with thesample; detecting an effect of the interaction of the reagent with thesample; providing output notifying the user of the results of thedetecting step. Examples of biochemical protocols for use with a dropletmicroactuator of the invention are discussed in the ensuing sections.

8.1 Nucleic Acid Amplification

The invention provides methods, devices and systems for amplification ofnucleic acids in droplets on a droplet microactuator. A large number ofcopies of one or more nucleic acid molecules can be made in a singledroplet from a small number of copies or even a single copy present inthe droplet. The methods of the invention generally involve combiningthe necessary reactants to form an amplification-ready droplet andthermal cycling the droplet at temperatures sufficient to result inamplification of a target nucleic acid, e.g., by the polymerase chainreaction (PCR). In some embodiments, a droplet including the amplifiedtarget nucleic acid may then be transported into a subsequent process,such as a detection process, a process for further manipulation of thetarget nucleic acid, and/or a sequencing process (e.g., as described inSection 8.2). Amplification devices may include a droplet microactuatorand components sufficient to conduct droplet operations affecting themethods of the invention when the droplet microactuator is loaded withappropriate reagents. Systems of the invention may include the dropletmicroactuator plus system components designed to facilitate softwarecontrol of the operation of the droplet microactuator to executeprotocols of the invention.

An illustrative droplet microactuator 100 for use in amplificationprotocols of the invention is illustrated in FIG. 1. In this embodiment,multiple fluid ports and/or reservoirs may be provided, such as samplereservoirs 102, PCR reagent reservoirs 104, and primer set reservoirs106. Heating areas may also be provided, such as lower temperatureheating area 108 and upper temperature heating area 110. A samplevisualization area 112 may also be provided, utilizing, for example, amicroscope or photomultiplier tube (PMT).

In one embodiment, the invention provides a droplet microactuator and/orsystem configured and programmed to effect amplification of a sample ina amplification-ready droplet followed by capture of the amplifiednucleic acid. The amplified nucleic acid may be treated to denature itinto single-stranded nucleic acid before or after it is contacted withmagnetically responsive beads to permit the single-stranded nucleic acidto bind to the magnetically responsive beads. Binding, for example, maybe accomplished using a biotin-streptavidin system, e.g., in which thesingle-stranded nucleic acid is biotinylated, and the surface (e.g.,beads or droplet microactuator surface) is coated with streptavidincovalently bound thereto. Amplification reagents may be washed awayusing a washing protocol. Various other methods, devices, systems, andother aspects of the invention will be apparent from the ensuingdiscussion.

It will be appreciated that an important aspect of the inventioninvolves the ability to conduct droplet operations using each of thenucleic acid amplification reagents and/or samples on a dropletmicroactuator. For example, the invention includes:

-   (1) a droplet microactuator comprising thereon a droplet comprising    any one or more of the reagents and/or samples described herein for    conducting nucleic acid amplification;-   (2) a device or system of the invention comprising such droplet    microactuator;-   (3) a method of conducting droplet operations on or otherwise    manipulating a droplet making use of such droplet microactuator or    system; and/or-   (4) a method of conducting an droplet-based sequence analysis    protocol making use of such droplet microactuator or system.

For example, the droplet operations may include one or more of thefollowing: loading a droplet into the droplet microactuator; dispensingone or more droplets from a source droplet; splitting, separating ordividing a droplet into two or more droplets; transporting a dropletfrom one location to another in any direction; merging or combining twoor more droplets into a single droplet; diluting a droplet; mixing adroplet; agitating a droplet; deforming a droplet; retaining a dropletin position; incubating a droplet; heating a droplet; vaporizing adroplet; cooling a droplet; disposing of a droplet; transporting adroplet out of a droplet microactuator; other droplet operationsdescribed herein; and/or any combination of the foregoing. Various othermethods, devices, systems, and other aspects of the invention will beapparent from the ensuing discussion.

8.1.1 Samples and Sample Preparation

The amplification methods of the invention make use of a sample whichincludes a nucleic acid template for amplification. The nucleic acidtemplate may be of any type, e.g., genomic DNA, RNA, plasmids,bacteriophages, and/or artificial sequences. The nucleic acid templatemay be from any source, e.g., whole organisms, organs, tissues, cells,organelles (e.g., chloroplasts, mitochondria), synthetic nucleic acidsources, etc. Further, templates may have a wide variety of origins,e.g., pathological samples, forensic samples, archaeological samples,etc. Biological specimens may, for example, include whole blood,lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum,cerebrospinal (CSF) fluids, amniotic fluid, seminal fluid, vaginalexcretions, serous fluid, synovial fluid, pericardial fluid, peritonealfluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine,gastric fluids, intestinal fluids, fecal samples, and swabs or washes(e.g., oral, nasopharangeal, optic, rectal, intestinal, vaginal,epidermal, etc.) and/or other biological specimens.

Various sample processing steps may be accomplished to prepare thenucleic acid template. In some cases, such as amplification fromplasmids or bacteriophages, crude sample will suffice. In other cases,such as amplification of large fragments from genomic DNA, highlypurified template is preferred. Sample preparation steps may take placeon or off the droplet microactuator.

The system of the invention may be configured and programmed to permitprocessing of a biological sample to prepare a droplet including anucleic acid template for amplification. Some portion or all of thisprocessing may be effected on or off the droplet microactuator, e.g.,using beads having reagents bound thereto with affinity for targetorganisms to isolate the target organisms from a biological sample. Thedroplet microactuator may process the sample by dividing it into one ormore discrete droplets for subsequent operations on the dropletmicroactuator.

Specimens may, in some instances, be treated to change reduce viscosityduring subsequent droplet operations. For example, samples can beprepared on the droplet microactuator or off the droplet microactuatorby mixing with an alkaline solution (for example, 10% KOH) or reducingagents such as dithiothreitol (DTT) or dithioerythritol (DTE) to liquefythe sample and render it sufficiently fluid to facilitate dropletoperations on a droplet microactuator. Other examples of suitable samplepreparation techniques are described in U.S. Patent Application No.60/745,950, entitled “Apparatus and Methods of Sample Preparation for aDroplet Microactuator,” filed on Apr. 28, 2006.

A droplet including the nucleic acid template may be combined withamplification reagents to provide an amplification-ready droplet, e.g.,combined with PCR reagents to yield a PCR-ready droplet. Depending onthe reagents selected, the amplification-ready droplet may beisothermally amplified or thermal cycled to effect amplification of atarget nucleic acid. Amplified product may be detected and/or quantifiedin real-time on a droplet microactuator. In this manner, the inventionprovides on-chip, real-time, quantitative amplification to detect andquantify a target nucleic acid in a sample.

As nearly 100% of the sample can be utilized for analysis (there is norequirement for priming of channels or pumps), very small sample volumes(e.g., less than about 100 μL or less than about 50 μL or less thanabout 25 μL) can be analyzed. Many tests can be performed using asingle, small sample, and reagent costs can be minimized.

8.1.2 Reagents

In the amplification protocols of the invention, various reagents may becombined with a nucleic acid template to yield an amplification-readydroplet, such as a PCR-ready droplet. PCR reagents typically includeprimers, nucleotides, polymerase, and buffers. These input reagents maybe provided as individual reagents loaded separately onto the dropletmicroactuator and combined using droplet operations on the dropletmicroactuator. Moreover, some or all of the reagents may be provided asreagent mixes that are loaded onto the droplet microactuator in apremixed form. In one embodiment, all amplification reagents arecombined into a single droplet that must only be combined with a sampledroplet in order to yield an amplification-ready droplet, e.g., aPCR-ready droplet.

8.1.2.1 Buffer

Reagents will typically include a buffer. The buffer is selected tofacilitate the amplification reaction. Any buffer which fulfills thisfunction is suitable. Magnesium ions are usefully included in the bufferwhere the nucleic acid being amplified is a DNA.

In one embodiment, the buffer includes KCl, Tris and MgCl₂. Othersuitable buffers are described in Chamberlain et al., Nucleic AcidResearch 16:11141-11156 (1988). For example, the buffer may compriseabout 500 mM KCl, about 100 mM Tris-HCl (pH 8.3), and about 15 mM MgCl₂.In another example, the buffer may comprise about 83 mM (NH₄)₂SO₄, about335 mM Tris-HCl (pH8.8), about 33.5 mM MgCl₂, about 50 mMβ-Mercapthoethanol, and about 34 mM EDTA. The buffer may also includeprimers and/or polymerases.

In one embodiment, PCR may be performed sequentially or in parallel inseveral droplets in which the concentration of one or more buffercomponents is systematically varied (e.g., in a series of droplets) inorder to improve or optimize the buffer for a specific reaction. Thus,for example, any one or more of the following buffer components may bevaried: KCl; Tris; MgCl₂; (NH₄)₂SO₄; β-Mercaptoethanol; EDTA. Once thebest of the tested buffer conditions is identified, PCR can proceedusing the best buffer system or further optimization may be conductedaround the best of the tested buffer systems.

The invention includes a droplet microactuator including a dropletthereon which is a buffer or which comprises a buffer component, as wellas systems and/or devices including such a droplet microactuator, andmethods of conducting droplet operations on or otherwise manipulatingsuch droplet on a droplet microactuator. Thus, for example, theinvention includes a droplet microactuator comprising a droplet thereon,which droplet comprises one or more of the following components: KCl,Tris, MgCl₂; (NH₄)₂SO₄; β-Mercapthoethanol; EDTA.

Further, the invention includes a droplet microactuator comprising adroplet thereon, which droplet comprises one or more of the foregoingcomponents at a concentration sufficient to facilitate amplification ofa target nucleic acid. Moreover, the invention includes such a dropletalong with a polymerase, nucleotides and/or one or more primers at aconcentration sufficient to facilitate amplification of a target nucleicacid. The invention also includes a method of conducting dropletoperations on or otherwise manipulating any of the droplets described inthis section using the droplet microactuator, device, and/or system. Forexample, the droplet operation may include one of more the following:loading a droplet into the droplet microactuator; dispensing one or moredroplets from a source droplet; splitting, separating or dividing adroplet into two or more droplets; transporting a droplet from onelocation to another in any direction; merging or combining two or moredroplets into a single droplet; diluting a droplet; mixing a droplet;agitating a droplet; deforming a droplet; retaining a droplet inposition; incubating a droplet; heating a droplet; vaporizing a droplet;cooling a droplet; disposing of a droplet; transporting a droplet out ofa droplet microactuator; other droplet operations described herein;and/or any combination of the foregoing.

8.1.2.2 Primers

Reagents used in the amplification methods of the invention will includeone or more primers. In typical methods, two primers are used to definethe region of the nucleic acid template that will be amplified. Primerswill typically have a sequence and a length which is selected to ensureefficient replication with few mistakes. Such primers are often in therange of about 18-24 bases long. Other requirements for selection ofeffective primers are known. Examples of suitable primer propertiesinclude lack of internal secondary structure, 40-60% G/C content,balanced distribution of G/C and A/T rich domains, and lack ofcomplementary at the 3′ ends to avoid formation of primer dimers. Thoughnot specifically required, primers with one or more of these propertiesmay be suitably employed in the practice of the invention. Additionally,the melting temperature (Tm) for primers is typically selected to permitannealing temperatures of about 55 to about 65° C., or about 62 to about65° C. A variety of publicly available computer programs exist to helpidentify primers with properties suitable for use in amplificationsettings. Where two primers are used, they are typically provided inequal concentrations. Primers may not be necessary in cases in which thenucleic acid being amplified is an RNA.

In some embodiments, degenerate mixtures of primers are used. Forexample, since a given amino acid sequence may be encoded by severalpossible codons, the mixture may include all possible sequences coveringall codon combinations for a target polypeptide. The degenerate primermixture may be simplified by identifying codon bias for the organism inquestion, and including only primers commonly used by the organism.

Primers are provided at any concentration which facilitatesamplification of the target nucleic acid. Concentrations should be lowenough to avoid an undue amount of mispriming, accumulation ofnon-specific product, and/or primer-dimer formation. Primerconcentration should be high enough to avoid exhaustion of primer priorto completion of the amplification reaction. In some embodiments,concentrations range from about 0.1 μM to about 1 μM or from about 0.1μM to about 0.6 μM.

Primers may also be labeled. For example, labels may be selected tofacilitate detection, localization, quantification, and/or isolation ofPCR product. For example, biotinylation can be used to facilitatedetection and/or purification using streptavidin to capture biotinylatedPCR product on surface. Further, streptavidin can be associated withmagnetically responsive beads for capture of biotinylated PCR product.Digoxigenin can also be used for detection of PCR product. Primers may,for example, be labeled at their 5′ ends and/or internally, and further,labeled nucleotides may be incorporated into the PCR product fordetection, localization, quantification, and/or isolation.

The invention includes a droplet microactuator including a dropletthereon which includes labeled and/or unlabeled primers (e.g., atconcentrations ranging from about 0.1 μM to about 1 μM or from about 0.1μM to about 0.6 μM) for amplification of a target nucleic acid in aconcentration sufficient to facilitate the amplification reaction, aswell as systems and/or devices including such a droplet microactuator,and methods of conducting droplet operations or otherwise manipulatingsuch droplet on a droplet microactuator. As another example, theinvention includes a droplet microactuator including a droplet thereonincluding labeled and/or unlabeled primers at a low enough concentrationto reduce or eliminate mispriming and accumulation of non-specificproduct and a high enough concentration to avoid exhaustion of primerprior to completion of the amplification reaction. In yet anotherexample, the invention includes a droplet microactuator comprising adroplet thereon including labeled and/or unlabeled primers at aconcentration ranging from about 0.1 μM to about 1 μM or from about 0.1μM to about 0.6 μM. Further, the invention includes such a droplet alongwith a polymerase, nucleotides and/or buffer components atconcentrations selected to facilitate amplification of a target nucleicacid. Moreover, the invention includes a method of conducting dropletoperations on or otherwise manipulating any of the droplets described inthis section using the droplet microactuator, device, and/or system. Forexample, the droplet operation may include one of more the following:loading a droplet into the droplet microactuator; dispensing one or moredroplets from a source droplet; splitting, separating or dividing adroplet into two or more droplets; transporting a droplet from onelocation to another in any direction; merging or combining two or moredroplets into a single droplet; diluting a droplet; mixing a droplet;agitating a droplet; deforming a droplet; retaining a droplet inposition; incubating a droplet; heating a droplet; vaporizing a droplet;cooling a droplet; disposing of a droplet; transporting a droplet out ofa droplet microactuator; other droplet operations described herein;and/or any combination of the foregoing.

8.1.2.3 Nucleotides

Reagents used in the amplification methods of the invention will includenucleotides. Stock solutions of dNTPs are commercially available from avariety of sources. Stock solutions are typically provided atconcentrations of 100-300 mM. Stock solutions can be diluted prior tointroduction to the droplet microactuator and/or on the dropletmicroactuator using droplet operations to combine the stock solutionswith one or more buffer solutions. Final concentrations in the PCR-readydroplet will typically range from about 50 μmol to about 200 μmol. Thefour dNTPs are typically provided in equimolar concentrations.

A variety of modified nucleotides may be employed in the practice of theinvention. Examples include nucleotides designed to reduce secondarystructure resolution, prevent contamination, as well as radiolabelednucleotides, non-radioactive labeled nucleotides, and nucleotidesdesigned to promote random mutagenesis. For examples, see McPherson etal., PCR, Taylor and Francis Group, 2006 (the entire disclosure which isincorporated herein by reference).

The invention includes a droplet microactuator including a dropletthereon which includes nucleotides for amplification of a target nucleicacid in a concentration sufficient to facilitate the amplificationreaction, as well as systems and/or devices including such a dropletmicroactuator, and methods of conducting droplet operations on orotherwise manipulating such a droplet on a droplet microactuator. Thus,for example, the invention includes a droplet microactuator comprising adroplet thereon including one or more nucleotides in a concentrationranging from about 100 mM to about 300 mM (stock concentration) or fromabout 50 μmol to about 200 μmol (operating concentration). In anotherexample, the invention includes a droplet microactuator comprising adroplet thereon including 1, 2, 3 or 4 nucleotides, each in aconcentration ranging from about 100 mM to about 300 mM or from about 50μM to about 200 μM. The system of the invention may be configured andprogrammed to execute a protocol for diluting stock nucleotideconcentrations to provide droplets comprising operating nucleotideconcentrations. For example, the system of the invention may beconfigured and programmed to execute a protocol diluting stocknucleotide concentrations ranging from about 100 mM to about 300 mM toprovide operating solutions ranging from about 50 μmol to about 200μmol. Further, the invention includes nucleotide-containing dropletsalong with polymerase(s), primer(s) and/or buffer components inconcentrations selected to provide conditions that facilitateamplification of a target nucleic acid. Moreover, the invention includesa method of conducting droplet operations on or otherwise manipulatingany of the droplets described in this section using the dropletmicroactuator, device, and/or system. For example, the droplet operationmay include one of more the following: loading a droplet into thedroplet microactuator; dispensing one or more droplets from a sourcedroplet; splitting, separating or dividing a droplet into two or moredroplets; transporting a droplet from one location to another in anydirection; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; cooling a droplet;disposing of a droplet; transporting a droplet out of a dropletmicroactuator; other droplet operations described herein; and/or anycombination of the foregoing.

8.1.2.4 PCR Polymerases

A variety of PCR polymerases may be used in the droplet-based PCRprotocols of the invention. Suitable polymerases will often have optimalactivity at about 75° C. and the ability to retain that activity afterprolonged incubation, e.g., at temperatures greater than 95° C. Usefulpolymerases may, for example, include DNA-dependent DNA polymerasesand/or RNA-dependent DNA polymerases (reverse transcriptases). Variousthermostable polymerases, such as Taq DNA polymerases, may be used.Suitable examples include AmpliTaq®, AmpliTaq Gold®, the Stoffelfragment of AmpliTaq®, and others. Examples of thermostable polymeraseswith proofreading capability include Vent®, Tli, DeepVent®, Pfu, Pwo,UlTma®, Accuzyme®, and KOD Hifi, DNA polymerases, as well as various exoversions of the foregoing. Polymerase preparations may in some casesinclude dyes for determining or confirming concentrations of PCRreagents. In some cases, the system is configured and programmed todetect such dyes and calculate reagent concentrations based oncolorimetric measurements. In some cases, the invention makes use ofdroplets including a combination of a thermostable polymerase (e.g., TaqDNA Polymerase) and a proofreading polymerase (e.g., Pwo DNAPolymerase).

The invention includes a droplet microactuator including a dropletthereon which includes one or more polymerases at concentrationssufficient to facilitate the amplification reaction, as well as systemsand/or devices including such a droplet microactuator, and methods ofconducting droplet operations or otherwise manipulating such droplet ona droplet microactuator. Moreover, the invention includes a method ofconducting droplet operations on or otherwise manipulating any of thedroplets described in this section using the droplet microactuator,device, and/or system. For example, the droplet operation may includeone of more the following: loading a droplet into the dropletmicroactuator; dispensing one or more droplets from a source droplet;splitting, separating or dividing a droplet into two or more droplets;transporting a droplet from one location to another in any direction;merging or combining two or more droplets into a single droplet;diluting a droplet; mixing a droplet; agitating a droplet; deforming adroplet; retaining a droplet in position; incubating a droplet; heatinga droplet; vaporizing a droplet; cooling a droplet; disposing of adroplet; transporting a droplet out of a droplet microactuator; otherdroplet operations described herein; and/or any combination of theforegoing. Further, the invention includes polymerase-containingdroplets on a droplet microactuator of the invention along withnucleotides, primer and/or buffer in concentrations selected to provideconditions sufficient to facilitate amplification of a target nucleicacid.

8.1.2.5 Detection of Amplified Product

In some embodiments, amplified nucleic acid will be detected after somenumber of amplification cycles. For example, amplified nucleic acid maybe quantified during or after each cycle, or after some predeterminednumber of cycles, e.g., after each set of 2, 3, 4, 5, 6, 7, 8, 9, or 10cycles. Detection generally involves using droplet operations totransport the droplet into detection zone in which a sensor measuressome aspect of the droplet, such as a physical, chemical or electricalaspect, which correlates with amplification. The system may beprogrammed so that amplification cycles may be continued until detectionreveals that a desired level of signal has been achieved. In oneembodiment, the detection method for amplification is a fluorescencetechnique.

Further, in some embodiments, a droplet comprising amplified nucleicacid may be transported for further processing/detection. For example,in diagnostic embodiments, a droplet including amplified nucleic acidmay be transported for detection of the presence of a target diagnosticnucleic acid. The target nucleic acid may, for example, include anucleic acid from the pathogenic organism, such as a DNA or RNAassociated with a parasite, bacteria, fungus or virus. The dropletmicroactuator may be provided as a component of an integrated andportable diagnostic platform. The system may provide for fullyautomated, rapid detection of a panel of infectious diseases byreal-time PCR. The system may be used at the bedside, stat laboratory,physician's office, or in the field.

Fluorescence detection is suitable for detection of amplified nucleicacid. Light emitting diodes (LEDS) and laser diodes are suitable asexcitation sources because of their small physical size, low powerrequirements and long life. LEDs are appealing because of their low costand laser diodes because of their narrow spectral width, and the factthat they can be focused to small spot sizes without discarding asubstantial amount of light.

In addition to the reagents already discussed, reagents usefullyemployed in nucleic acid amplification protocols of the inventioninclude various detection reagents, such as fluorescent andnon-fluorescent dyes and probes. For example, the protocols may employreagents suitable for use in a TaqMan reaction, such as a TaqMan probe;reagents suitable for use in a SYBR® Green reaction, such as SYBR®Green; reagents suitable for use in a molecular beacon reaction, such asa molecular beacon probe; reagents suitable for use in a scorpionreaction, such as a scorpion probe; reagents suitable for use in afluorescent DNA-binding dye-type reaction, such as a fluorescent probe;and/or reagents for use in a LightUp protocol, such as a LightUp probe.

The invention includes a droplet microactuator including a dropletthereon which includes one or more detection reagents, such as any oneor more of the aforementioned probes, at concentrations sufficient tofacilitate detection of the amplification reaction, as well as systemsand/or devices including such a droplet microactuator, and methods ofconducting droplet operations or otherwise manipulating such droplet ona droplet microactuator. Moreover, the invention includes a method ofconducting droplet operations on or otherwise manipulating any of thedroplets described in this section using the droplet microactuator,device, and/or system. For example, the droplet operation may includeone of more the following: loading a droplet into the dropletmicroactuator; dispensing one or more droplets from a source droplet;splitting, separating or dividing a droplet into two or more droplets;transporting a droplet from one location to another in any direction;merging or combining two or more droplets into a single droplet;diluting a droplet; mixing a droplet; agitating a droplet; deforming adroplet; retaining a droplet in position; incubating a droplet; heatinga droplet; vaporizing a droplet; cooling a droplet; disposing of adroplet; transporting a droplet out of a droplet microactuator; otherdroplet operations described herein; and/or any combination of theforegoing. Further, the invention includes nucleotide-containingdroplets on a droplet microactuator of the invention along with anucleotides, primer, detection probe and/or buffer in concentrationsselected to provide conditions sufficient to facilitate amplification ofa target nucleic acid.

Furthermore, the invention includes methods of detecting and/orquantifying amplification which methods include measuring a signal(e.g., a fluorescent signal) from droplet on a droplet microactuator.Thus, for example, the method may employ exposure of a dropletpotentially including a fluorescent dye (such as SYBR® Green) to a lightsource at a wavelength selected to cause the compound to fluoresce andmeasuring the resulting fluorescence. Fluorescence emitted from thedroplet can be tracked during an amplification reaction to permitmonitoring of the reaction, e.g., using a SYBR® Green-type compound. Asystem of the invention may, for example, be programmed to detect suchfluorescence and to display a corresponding graph or other outputpermitting a user to track the progress of a PCR reaction in real-time.

In another example, the invention includes a method of detecting and/orquantifying the presence of a target nucleic acid by including a probewith specificity for a target nucleic acid (e.g., a TaqMan-type probe)in an amplification-ready droplet potentially including the targetnucleic acid, thermal cycling the amplification-ready droplet, anddetecting any fluorescence signal caused by degradation of the probe,where a fluorescent signal is indicative of the presence of the targetnucleic acid in the droplet. The invention includes correspondingmethods using other target-specific probes, such as scorpion probes andmolecular beacons.

8.1.3 Thermal Cycling

In the practice of the invention, a PCR-ready droplet is thermal cycledin order to effect an amplification of a target nucleic acid. Tightcontrol of thermal cycling may be necessary for effective amplificationof certain nucleic acids. Examples of structures designed to providecontrolled thermal cycling on a droplet microactuator are discussed inSection 8.8.6 below. Typically, each thermal cycle will involve at leasttwo steps:

-   (1) heating the droplet to a temperature sufficient to denature    double stranded nucleic acid in the droplet into single-stranded DNA    (typically about 90-100° C.); and-   (2) lowering the droplet temperature to permit primers to anneal to    their complementary sequences on the nucleic acid template strands    (typically about 50-75° C.).

In some cases a thermal cycle may also involve a third step:

-   (3) adjusting the droplet temperature to facilitate extension of the    double stranded segment of the nucleic acid to be extended by    incorporation of additional nucleotides (typically about 70-75° C.).

Depending on the reagents selected, incorporation of additionalnucleotides may be accomplished at the same temperature at which theprimers are permitted to anneal to the nucleic acid template strands,and thus the temperature adjustment of the third step may not benecessary. Additional thermal cycling steps may also be incorporated invarious protocols of the invention.

The invention permits multiple droplets to be thermal cycled inparallel. Thus, in various embodiments, more than 2, 3, 4, 5, 10, 20,30, 40, 50, 100, or 1000 amplification-ready droplets are thermal cycledin parallel on a single droplet microactuator. In some embodiments,detection of amplification in these droplets is measured in parallel inreal time.

In one embodiment, each droplet undergoing thermal cycling is positionedin proximity to a sensor, or is transported into proximity with asensor, so that a signal from the droplet correlating with amplificationcan be monitored in real time. The system may output real-timeinformation accessible to a user which is indicative of the progress ofthe amplification process. Further, the system may be arranged to permitsuch output when thermal cycling multiple droplets in parallel, e.g.,more than 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, or 1000amplification-ready droplets are thermal cycled in parallel on a singledroplet microactuator and the system outputs real-time information isindicative of the progress of the amplification process

The methods of the invention may include a temperature optimization stepor protocol for optimizing temperatures and/or times for denaturation,annealing, and/or extension. In this step, one or more heating zones isused to vary the temperature of one of more heating steps.

For example, the methods may include an optimization step or protocolfor optimization of annealing temperature. A series of droplets may bethermal cycled using varied annealing temperatures followed by detectionto quantify amplification and thereby determine which of the testedannealing temperatures produces the best results. Subsequent thermalcycling can be conducted at the selected temperature. Similarly a seriesof droplets may be cycled through annealing temperatures for differentperiods of time followed by detection to quantify amplification andthereby determine which of the tested time periods produces the bestresults at a given temperature. Subsequent thermal cycling can beconducted using the selected time period. Further, such optimizationprotocols can be executed sequentially or simultaneously in order todetermine both the optimum temperature and the optimum time period.Similar protocols may be executed for optimizing temperatures and/ortime periods for denaturation and/or extension steps.

In one embodiment, thermal cycling is accomplished by heating andcooling the entire droplet microactuator. This embodiment generallyinvolves the following steps:

-   (1) heating the droplet microactuator to a temperature sufficient to    denature the double-stranded DNA (present in a droplet on the    droplet microactuator) into single-stranded DNA;-   (2) lowering the temperature of the droplet microactuator to a    temperature sufficient to permit primers (present in a droplet on    the droplet microactuator) to anneal to their complementary    sequences on the nucleic acid template strands;-   (3) optionally, adjusting the temperature of the droplet    microactuator to facilitate extension of the double stranded segment    of the nucleic acid (present in a droplet on the droplet    microactuator) by incorporation of additional nucleotides.

The thermal cycling protocols of the invention may be conducted withoutsignificant loss of water or other components of the PCR-ready droplet.Further, the thermal cycling protocols may be conducted withoutsignificant cross-contamination between droplets. Moreover, the thermalcycling may be conducted without significant disruption in thecapability of the droplet microactuator to continue conducting dropletoperations. For example, droplet operations may in some cases continueto be conducted at the various denaturation, annealing, and/or extensiontemperatures.

In a related embodiment, thermal cycling is accomplished by heating andcooling a section or region of the droplet microactuator. This approachgenerally involves the following steps:

-   (1) heating a section or region of the droplet microactuator to a    temperature sufficient to denature the double-stranded DNA (present    in a droplet on the droplet microactuator) into single-stranded DNA;-   (2) lowering the temperature of a section or region of the droplet    microactuator to a temperature sufficient to permit primers (present    in a droplet on the droplet microactuator) to anneal to their    complementary sequences on the nucleic acid template strands;-   (3) optionally, adjusting the temperature of a section or region of    the droplet microactuator to facilitate extension of the double    stranded segment of the nucleic acid (present in a droplet on the    droplet microactuator) by incorporation of additional nucleotides.

In one embodiment, this approach is conducted using a dropletmicroactuator with a single integrated heater 202, as illustrated inFIG. 2A.

In another embodiment, regions of the droplet microactuator may bemaintained at the required temperatures, and the droplets may betransported through the appropriate temperature regions in order toaccomplish the thermal cycling. This approach generally involves thefollowing steps:

-   (1) transporting an amplification-ready droplet to a region of the    droplet microactuator that is maintained at a temperature    appropriate to cause denaturation of double-stranded DNA in the    droplet into single-stranded DNA;-   (2) transporting the amplification-ready droplet to a region of the    droplet microactuator that is maintained at a temperature sufficient    to permit primers in the droplet to anneal to their complementary    sequences on the nucleic acid template strands; and-   (3) optionally, transporting an amplification-ready droplet to a    region of the droplet microactuator that is maintained at a    temperature sufficient to facilitate or optimize extension of the    double-stranded segment of the nucleic acid in the droplet by    incorporation of additional nucleotides.

In this embodiment, thermal cycling is accomplished by repeating thetransporting steps to move the droplets from zone to zone. In oneembodiment, the droplet microactuator includes only one thermal zone foreach required temperature, and thermal cycling is accomplished byrotating each droplet through the appropriate thermal zones. In anotherembodiment, the droplet microactuator includes two or more of each ofthe thermal zones. In yet another embodiment, the droplet microactuatorincludes two or more of one or more of the thermal zones. Further, thedroplet microactuator may include 2, 3 or more thermal zones, each ofwhich may be heated to a different specified temperature. In oneembodiment, this approach is conducted using a droplet microactuatorwith a plurality of integrated heaters 204, as illustrated in FIG. 2B.

Further, one or more heaters may be used to establish a continuousthermal gradient across a region of the droplet microactuator. In thisembodiment, an electrode matrix, electrode path or series of electrodepaths may be employed to locate the droplet in the appropriatetemperature zone to effect the required thermal cycling steps. Thermalcycling may thus be accomplished by transporting the droplet to anelectrode at the appropriate position within the thermal gradient toachieve the target temperature. Variations in temperature, e.g., tooptimize any of the various denaturing, annealing and/or extensionsteps, may be effected by simply varying the location of the dropletwithin the thermal zone.

Thermal cycling may involve the use of various heating and/or coolingmodalities to establish target temperature zones for denaturation,annealing and/or extension. These heating and cooling modalities may bearranged to facilitate a suitable temperature ramp between the targettemperature zones. The ramp may be controlled by changing thetemperature of a specific heating and/or cooling modality and/or byselecting heating and cooling modalities at distances selected to effecttarget temperature zones with suitable temperature ramps. The dropletmicroactuator may have heating and/or cooling modalities withtemperature ranges and spacing selected to create a predetermined set ofpotential target temperature zones and temperature ramps. Variousheating and/or cooling modalities may be included between targettemperature zones to adjust the ramp between zones.

In one embodiment, the droplet microactuator includes a series ofindependently adjustable heating elements. The temperature of eachheating element may be adjusted to provide for an appropriate heatingramp as droplets pass from one target temperature zone to the next.Further, distance between heating elements at target temperatures may beselected to facilitate an appropriate temperature ramp and/or to preventoverheating caused by interaction between closely situated heatingelements. Such approaches provide flexibility in working with a varietyof protocols each requiring different target temperature zones and rampprofiles. For example, in a series or matrix of heating elements, targettemperature zones may be at adjacent heating elements and/or may beseparated by one or more heating elements such that they are separatedby a greater distance. In this way, distances may be varied to accountfor temperature requirements of a variety of protocol requirements. Asystem of the invention may select optimal heating elements forestablishing target temperature zones with appropriate or optimaltemperature ramps between the heating zones.

The methods of the invention may include a temperature optimization stepor protocol for optimizing temperatures and/or times for thedenaturation, annealing, and/or extension phases of the thermal cycle.As an example, the methods may include an optimization step or protocolfor optimization of annealing temperature. A series of heating zones maybe established in which amplification-ready droplets are cycled throughdifferent annealing temperatures to determine which annealingtemperature produces optimum results. Subsequent thermal cycling can beconducted at the optimum temperature. Similarly a series ofamplification-ready droplets may be cycled using a protocol in which thetime of the annealing phase is systematically varied to determine whichtime period produces optimum results at a given temperature. Subsequentthermal cycling can be conducted using the optimum time period. Further,such protocols can be executed sequentially or simultaneously in orderto determine both the optimum temperature and the optimum time period.Similar protocols may be executed for optimizing denaturation and/orextension steps. Optimization protocols my run sequentially or inparallel.

Similarly, the methods of the invention may include a temperatureoptimization step or protocol using multiple independently heatedthermal zones for optimizing temperatures and/or times for denaturation,annealing, and/or extension. For example, a series of heating zones maybe established through which amplification-ready droplets aretransported. The zones may include temperatures targeted to promotedenaturation, annealing, and/or extension. In a specific droplet set,the temperature of one of the denaturation, annealing and/or extensionzones may be systematically varied for a set of PCR-ready droplets,while the other two temperatures remain constant. Multiple droplet setscan be tested so that each of the temperature parameters may be variedas needed. One or more of the multiple droplet sets may be testedsequentially and/or in parallel. Variations in heating zone temperaturemay, for example, be controlled by the processor (e.g., controlling thetemperature of a heating element and/or controlling a location of adroplet within a heating gradient) via software pre-programmed toexecute an optimization protocol and/or via software controlled by auser via a user interface. The timing of each of the phases of thethermal cycle may be optimized in like manner. Optimization protocols myrun sequentially or in parallel.

Moreover, the invention includes a method of conducting one or moredroplet operations using a PCR-ready droplet on a droplet microactuatorat an elevated temperature, e.g., at a temperature which is greater thanabout 70, 75, 80, 85, 90, 95, or 100° C. For example, the dropletoperation may include: loading a droplet into the droplet microactuator;dispensing one or more droplets from a source droplet; splitting,separating or dividing a droplet into two or more droplets; transportinga droplet from one location to another in any direction; merging orcombining two or more droplets into a single droplet; diluting adroplet; mixing a droplet; agitating a droplet; deforming a droplet;retaining a droplet in position; incubating a droplet; heating adroplet; vaporizing a droplet; cooling a droplet; disposing of adroplet; transporting a droplet out of a droplet microactuator; otherdroplet operations described herein; and/or any combination of theforegoing. Still further, the invention includes a method of heatingand/or cooling a droplet by transporting the droplet between two or moretemperature zones on a droplet microactuator. Further, the inventionincludes a method of heating and/or cooling a droplet by transportingthe droplet between two or more temperature zones on a dropletmicroactuator when the temperature zones range from about 40° C. toabout 120° C. The invention also includes a method of heating and/orcooling a droplet by transporting the droplet between two or moretemperature zones on a droplet microactuator when the temperature zonesrange from about 40° C. to about 120° C. to achieve target temperaturesat least some of which are at a temperature which is greater than about70, 75, 80, 85, 90, 95, or 100° C.

The invention includes a droplet microactuator or droplet microactuatorsystem having one or more input reservoirs loaded with reagents forconducting biochemical reactions, such as the reagents described for usein nucleic acid amplification protocols, affinity-based assay protocols,sequencing protocols, and protocols for analyses of biological fluids.For example, one or more reservoirs may include reagents for providingbuffer, primers, nucleotides, polymerases, and other reagents forconducting a PCR reaction. In one embodiment, one or more reservoirsincludes a buffer which includes two or more reagents for conducting aPCR reaction, where in the reagents are selected from primers,nucleotides, polymerases, and other PCR reagents. In another embodiment,one or more of the reservoirs includes a droplet including all reagentsrequired for conducting a PCR reaction, such that when combined with asample droplet including a nucleic acid template, the result is adroplet which is ready for PCR thermal cycling. The invention alsoincludes a droplet microactuator or droplet microactuator system, havingone or more input reservoirs loaded with a sample for conducting a PCRreaction.

8.1.4 Amplification Protocols

It will be appreciated by one of skill in the art in light of thepresent disclosure that many variations are possible within the scope ofthe invention. In general, the protocols involve combining two or moredroplets comprising PCR reagents and template to yield a PCR-readydroplet, and thermal cycling the PCR-ready droplet at temperaturesselected to facilitate amplification of a target nucleic acid.

Upstream, the protocol may involve various sample processing steps inorder to provide a nucleic acid template that is ready for PCRamplification. For example, reverse transcription of RNA may be usedprior to PCR to provide a stable DNA nucleic acid template foramplification. Thus, in one embodiment, the invention provides a methodof preparing a droplet comprising a DNA nucleic acid template, whereinthe method includes performing droplet-based reverse transcription ofRNA to yield the nucleic acid template.

A “Hot Start” approach can be used to minimize the formation ofprimer-dimers during reaction preparation. By limiting polymeraseactivity prior to PCR cycling, non-specific amplification is reduced andtarget yield is increased. Common methods for Hot Start PCR includechemical modifications, wax-barrier methods, and inhibition by aTaq-directed antibody.

Downstream, the protocol may involve various subsequent steps, such assequencing of an amplified nucleic acid, e.g., using a pyrosequencingapproach or separation of amplified fragments using capillaryelectrophoresis,

Various specialized techniques may also be used during the PCR process.For example, primers with sequences not completely complementary to thenucleic acid template can be used for droplet-based in vitromutagenesis. Thus, for example, the invention may include a method ofaccomplishing in vitro mutagenesis in a droplet on a dropletmicroactuator, when the method involves combining two or more dropletsincluding PCR reagents and primers selected for mutagenesis inquantities sufficient to facilitate amplification of a mutated versionof the target nucleic acid. Further, the mutated version of the targetnucleic acid may be transported for downstream processing, e.g.,sequencing of the mutated version of the target nucleic acid to confirmthe desired mutation.

In a medical diagnostic aspect of the invention, molecular tags, such asdigoxigenin (DIG) or biotin-labeled dUTP can be used to permit detectionof specific sequences. The labeled PCR products may, for example, beused as hybridization probes or detected by use of capture probes.

In many protocols, it will be desirable to simultaneously process one ormore control droplets to determine the quality or fidelity of thereaction. Thus, for example, in order to ensure that contamination hasnot occurred one or more PCR-ready droplets without the sample templatemay be thermal cycled and otherwise processed in the same manner asdroplets including the sample template. Detection of amplified nucleicacid in the control droplets would be indicative of contamination. Othercontrol droplets may include known quantities or concentrations oftemplate material, or known quantities or concentrations of fluorescentdye.

Control droplets may be processed on the same droplet microactuator asthe sample droplets, simultaneously with, before and/or after,processing of the actual sample droplet.

The system provides the possibility of independent software-basedcustomization of reaction protocols and conditions for each sample orassay. This, combined with the scalability of the platform ensures thatthe capabilities of the system can be extended to include a wide rangeof nucleic acid targets.

The invention includes methods of conducting droplet operations onamplification reagents. For example, the invention includes a method ofconducting one or more droplet operations on a droplet including buffer,primers, nucleotides, polymerases, and/or other PCR reagents.

The invention also includes a method of conducting one or more dropletoperations using a buffer droplet on a droplet microactuator includingone or more primers. The invention also includes a method of conductingone or more droplet operations using a buffer droplet on a dropletmicroactuator including one or more nucleotides. The invention alsoincludes a method of conducting one or more droplet operations using abuffer droplet on a droplet microactuator including one or morepolymerases, e.g., DNA polymerases. The invention also includes a methodof conducting one or more droplet operations using a buffer droplet on adroplet microactuator including one of more reverse transcriptases. Inanother embodiment, the invention includes a method of conducting one ormore droplet operations using a buffer droplet on a dropletmicroactuator which includes 2, 3, 4 or more PCR reagents selected fromthe categories including primers, nucleotides, polymerases, and otherPCR reagents. Further, the invention includes a method of conducting oneor more droplet operations using a PCR-ready droplet including one ormore buffers, primers, nucleotides, polymerases, and nucleic acidtemplates including a target nucleic acid sequence.

The droplet-based amplification protocols are also useful for analyzingRNA content. In some embodiments, RNA will be the initial target nucleicacid. A two buffer system may be used to provide one buffer for thereverse transcription (RT) step that creates cDNA from the viral RNA,and a different buffer selected to facilitate the DNA amplificationstep. In a related embodiment, a single buffer method is used in which abuffer is provided that is compatible for both reactions but notnecessarily optimal for either.

In one embodiment, a droplet-based PCR can be executed on a dropletmicroactuator to quantitate the changes in gene expression levels forrelevant cancer biomarkers, e.g., vascular endothelial growth factor(VEGF) and the cyclin-dependent kinase inhibitors p21(Cip1) andp27(Kip1). For example, cells in a droplet, whether suspended or boundto a surface, can be lysed. Freed poly(A) mRNA can be captured indroplets using beads, such as oligo (dT) magnetically responsive beads.Reagents are available from Dynal Biotech in its mRNA DIRECT Micro Kit.Mixing or agitation of droplets may be used to enhance cell lysis andenhance capture of poly (A) mRNA onto beads. mRNA from oligo (dT)magnetically responsive beads can be eluted by thermally melting theRNA-DNA duplex. The appropriate temperature depends on the length of thestrand. Beads can be washed using droplet-based washing protocols asdescribed elsewhere herein. PCR (e.g., qRT-PCR) can be performed using adroplet-based protocol on the droplet microactuator with the appropriateprimers for the gene targets (e.g., VEGF, p21(Cip1) and p27(Kip1)).Droplet-based RNA amplification may also be accomplished using the VanGelder and Eberwine technique.

The invention provides droplet-based rolling circle amplification forDNA. In the rolling circle approach, a buffer droplet including a dsDNAhis heated on a droplet microactuator to a temperature sufficient toresult in denaturation of the dsDNA (typically about 95° C.). Incubationtime may in some instances range from about 1 to about 10 minutes. Adroplet including a circularizable probe is combined with the dropletincluding the denatured DNA to anneal and ligate the circularizableprobe to the target dsDNA at an effective temperature (e.g., about 60°C.) in buffer with a polymerase (e.g., T. flavus DNA polymerase) and anappropriate ligase (e.g., Ampligase DNA ligase). Incubation may in somecases be less than about 45 minutes. The resulting droplet is combinedwith rolling circle primer, buffer, 029 DNA polymerase, at a decreasedtemperature (e.g., about 31° C.). Incubation time may in some casesrange from about 2 to about 30 minutes. Biotin may be incorporated withthe 029 DNA polymerase to capture the amplicon on a streptavidin bead orsurface and visualized with a fluorescent probe.

The invention provides droplet-based strand displacement amplification(SDA) for DNA. In this embodiment, a buffer droplet including a dsDNAfragment with target specific amplification primers is heated on adroplet microactuator to a temperature sufficient to result indenaturation of the dsDNA (typically about 95° C.). Incubation time mayin some instances be for less than about 4 minutes. The droplet is thencooled to an annealing temperature (e.g., about 37° C.) to result inannealing. Annealing time may in some cases be for less than about 4minutes. The droplet is combined using droplet operations with a dropletincluding a restriction endonuclease and exo(minus) Klenow polymerase.The resulting droplet is isothermally incubated on the dropletmicroactuator at a temperature sufficient to result in DNA amplification(e.g., about 37° C.). Incubation time may in some cases be from about 1to about 5 hours. Amplification can be detected using, for example, afluorescent probe or a strand specific molecular beacon

The invention provides droplet-based transcription mediatedamplification or NASBA for RNA. In this embodiment, a droplet includinga target nucleic acid is heated on the droplet microactuator to atemperature sufficient to denature the target (e.g., about 95° C.).Denaturation time may in some cases be less than about 4, 3, or 2minutes. The droplet is then cooled to an appropriate temperature (e.g.,about 41° C.), and combined using droplet operations with a dropletincluding add T7 RNA polymerase promoter-target primer 1 and targetprimer 2. The resulting droplet is combined using droplet operationswith a droplet including reverse transcriptase, RNAse H and T7 RNApolymerase. The droplet temperature is then adjusted to a temperaturesufficient to result in amplification of RNA amplicons. Amplificationtime may in some cases last for about 60 minutes.

One aspect of the invention is a droplet microactuator having asubstrate for immobilization of a nucleic acid. In one aspect, thesubstrate is a gold substrate. Another aspect is a droplet microactuatorincluding a substrate for immobilization of a nucleic acid and reagentsfor immobilizing the nucleic acid to the substrate. Yet another aspectis a droplet microactuator including a substrate for immobilization of anucleic acid, reagents for immobilizing the nucleic acid to thesubstrate, and a nucleic acid sample. These reagents and samples, mayfor example, be stored in reservoirs on the droplet microactuator and/orin reservoirs or other containers off the droplet microactuator (e.g.,in a cartridge). In yet another aspect, the invention involves a methodof immobilizing a nucleic acid sample on a substrate comprisingexecuting droplet operations to bring a droplet comprising the nucleicacid sample into contact with the substrate and thereby deposit in thenucleic acid sample on the substrate.

8.1.5 Downstream Analysis

In some embodiments, a droplet comprising amplified target nucleic acidmay be transported downstream for further analysis. For example, thedroplet may be transported and stream for analysis by micro gelelectrophoresis. The micro gel electrophoresis may take place on or offthe droplet microactuator.

In one embodiment, a two dimensional micro gel electrophoresis device,such as the device described by Mohanty et al. and the AmericanElectrophoresis Society Annual Meeting (seehttp://aiche.confex.com/aiche/2005/techprogram/P30621.HTM).

In some embodiments, droplets including amplified nucleic acids arecontacted with droplets including reagents sufficient to clone theamplified nucleic acids into suitable vectors. Vectors may be selected,for example, for use in gene libraries, and/or expression in cells.

8.2 Nucleic Acid Sequence Analysis

The invention provides methods, devices and systems for droplet-basednucleic acid sequence analysis on a droplet microactuator system which,among other things, avoids problems associated with the increasinglycomplex mixtures required by the approaches of the prior art.

FIG. 3 illustrates an illustrative droplet microactuator 300 suitablefor nucleic acid sequence analysis. In this embodiment, multiple fluidports or reservoirs may be provided such as DNA input reservoir 302, DNAreagents reservoir 304, primer set reservoirs 306, nucleotide (e.g., dA,dC, dG, and dT) reservoirs 308, and pyrosequencing primer reservoir 310.Wash buffer reservoir 312 may also be provided, as well as waste area314, thermal cycling area 316, and imaging area 318. Various thermalcycling area embodiments may employ a variety of heater configurationssuch as those described elsewhere herein. Imaging area 318 may utilize,for example, a photomultiplier tube (PMT).

FIGS. 4 and 5 illustrate reaction steps and droplet operations of anillustrative embodiment of the invention. A nucleic acid sample may beamplified as needed (on or off the droplet microactuator) to obtain asufficient concentration of nucleic acid for analysis. The nucleic acidsample may be introduced to a droplet microactuator where it isimmobilized on a solid support. Reagents for denaturing the nucleic acidto single strand, priming and stepwise extension of the double strandedportion, may be transported to the immobilized nucleic acid usingdroplet microactuation techniques. Importantly, droplets includingreaction products may be transported away from the immobilized nucleicacid, e.g., for further processing, analysis, and/or waste disposal.Importantly, detection may in some embodiments be conducted separatelyin time and space relative to the extension synthesis reactions. Amongother things, this capability reduces or eliminates the build-up ofcertain degradation byproducts caused by existing methods. A furtheradvantage of the invention is that detection can occur in proximity to asensor to improve the efficiency of light collection and thus thesensitivity of the analysis.

The invention may include a droplet microactuator or dropletmicroactuator system having one or more input reservoirs loaded withreagents for conducting sequencing protocols. For example, one or morereservoirs may include reagents for conducting a pyrosequencingprotocol. The invention also may include a droplet microactuator ordroplet microactuator system, having one or more input reservoirs loadedwith a sample for conducting a pyrosequencing protocol.

It will be appreciated that an important aspect of the inventioninvolves the ability to conduct droplet operations using each of thesequence analysis reagents and/or samples on a droplet microactuator.For example, the invention may include:

-   (1) a droplet microactuator comprising thereon a droplet comprising    any one or more of the reagents and/or samples described herein for    conducting sequence analyses;-   (2) a device or system of the invention comprising such droplet    microactuator;-   (3) a method of conducting droplet operations on or otherwise    manipulating a droplet making use of such droplet microactuator or    system; and/or-   (4) a method of conducting an droplet-based sequence analysis    protocol making use of such droplet microactuator or system.

For example, the droplet operations may include one or more of thefollowing: loading a droplet into the droplet microactuator; dispensingone or more droplets from a source droplet; splitting, separating ordividing a droplet into two or more droplets; transporting a dropletfrom one location to another in any direction; merging or combining twoor more droplets into a single droplet; diluting a droplet; mixing adroplet; agitating a droplet; deforming a droplet; retaining a dropletin position; incubating a droplet; heating a droplet; vaporizing adroplet; cooling a droplet; disposing of a droplet; transporting adroplet out of a droplet microactuator; other droplet operationsdescribed herein; and/or any combination of the foregoing. Various othermethods, devices, systems, and other aspects of the invention will beapparent from the ensuing discussion.

8.2.1 Sample Amplification

Nucleic acid sequence analysis typically begins with a sample includingan amplified nucleic acid analyte or with a sample that includes anucleic acid analyte for amplification. For the latter, amplificationcan be performed using standard techniques and/or using droplet-basedamplification on a droplet microactuator as described in Section 8.1.Amplification may be conducted on the same droplet microactuator used toconduct sequence analysis protocols and/or on a separate dropletmicroactuator. In some embodiments, a second droplet microactuator andis coupled in fluid communication with a sequence analysis dropletmicroactuator.

8.2.2 Nucleic Acid Immobilization

As illustrated in FIG. 4, the amplified nucleic acid sample may beimmobilized within the droplet microactuator so that reagent dropletsmay be brought into contact with the immobilized nucleic acid.Immobilization may be on a surface of the droplet microactuator or onother surfaces within the microactuator, such as beads made frompolymers, polymeric resins, and optionally including magneticallyresponsive materials.

Useful substrates for such attachment include glass, gold,polyacrylamide gels, polypyrrole, Teflon, and optical fibers. Materialsmay, for example, be provided as films, particles, matrices or beads. Inone embodiment, the substrate includes magnetically responsive beads.

The droplet microactuator can include a magnet or electromagnet forproducing a magnetic field to manipulate (e.g., immobilize, release, ormove) the magnetically responsive beads. For example, magneticallyresponsive beads can be agitated and immobilized on the microactuatorusing a magnetic field to enhance washing steps (see Section 8.6).

A wide variety of techniques may be used to immobilize molecules, suchas DNA, to surfaces. Examples include those chemistries used to attacholigonucleotides to the surface of microarrays and chemistries used insolid phase synthesis techniques.

Nucleic acid samples may be thiolated and adsorbed to a gold substrate.Nucleic acids maybe thiolated, for example, by substituting anon-bridging internucleotide oxygen of a phosphodiester moiety withsulfur, e.g., as described in U.S. Pat. No. 5,472,881, by Beebe et al.,entitled “Thiol Labeling of DNA for Attachment to Gold Surfaces,” theentire disclosure of which is incorporated herein by reference. One ormore droplets including the thiolated nucleic acid can be transported onthe droplet microactuator to a gold surface where the thiolated nucleicacid sample will be deposited on the gold surface. The droplet includingthe thiolated DNA is brought into contact with the gold surface for asufficient time for covalent bonds to form between the sulfur and thegold. Electroactuation techniques may be employed to increase thesurface area of the droplet with the gold surface.

DNA sample can be biotinylated at the 5′-ends using a water solublebiotin ester or using a biotinyl phosphoramidite reagent. BiotinylatedDNA can be captured on streptavidin coated substrates. Thus, a dropletincluding biotinylated DNA sample can be transported into contact withthe streptavidin surface where the DNA will be captured and immobilized.

Chemistry has been described for immobilization of single stranded DNAon a substrate (S. Taira et al., “Immobilization of single-stranded DNAby self-assembled polymer on gold substrate for a DNA chip,” BiotechnolBioeng. 2005 Mar. 30; 89(7):835-8). In this approach, thioctic acid (TA)is covalently attached to poly(allylamine hydrochloride) (PAH) insidechains to immobilize the polymer on a gold surface by self-assembly.N-hydroxysuccinimide-ester terminated probe single-stranded (ss) DNA iseasily covalently immobilized onto a TA-PAH-coated gold surface. Thesurface may be covered with polyacrylic acid, which forms ion complexeswith the TA-PAH, to reduce the cationic charge.

As illustrated in step 3 of FIG. 4, double stranded nucleic acid istreated with a denaturing reagent, such as NaOH solution, in order toyield single stranded sample. This step is illustrated as occurringafter the immobilization step; however, it will be appreciated thatdenaturation may be effected by transporting a denaturing reagent intocontact with the double stranded nucleic acid sample, before, during orafter immobilization. Denaturation may also be performed by heating thesample to thermally melt the double-stranded complex.

One aspect of the invention is a droplet microactuator having asubstrate for immobilization of a nucleic acid. Another aspect is adroplet microactuator including a substrate for immobilization of anucleic acid and reagents for immobilizing the nucleic acid to thesubstrate. Yet another aspect is a droplet microactuator including asubstrate for immobilization of a nucleic acid, reagents forimmobilizing the nucleic acid to the substrate, and a nucleic acidsample. These reagents and samples, may for example, be stored inreservoirs on the droplet microactuator and/or in reservoirs or othercontainers off the droplet microactuator (e.g., in a cartridge). In oneembodiment of a reservoir loading assembly, reagent and/or samplereservoirs (e.g., in vials or syringes) may be coupled in fluidcommunication with droplet microactuator reservoirs so that fluid fromthe vials may flow or be forced directly into the droplet microactuatorreservoirs. This aspect of the invention is scalable, such that thenumber of reservoirs and reservoir loading assemblies may be increasedas needed to include slots for as many reagents as required to conduct adesired protocol. Reagent/sample reservoirs and reagent/sample loadingare discussed further in Sections 8.8.4 and 8.8.5.1.

8.2.3 Polymerase Facilitated Nucleotide Incorporation

As illustrated in steps 4 and 5 of FIG. 4, the immobilized, singlestranded nucleotide sample is primed to yield a double stranded segment.Priming may, for example, be achieved by transporting a dropletcomprising primer into contact with the immobilized sample.

The primed sample is reacted with a deoxynucleotide triphosphate (dNTP)in the presence of a polymerase. This reaction may be achieved bytransporting one or more droplets including a deoxynucleotidetriphosphate (dNTP) and a polymerase into contact with the immobilizedsample. If the dNTP is complementary to the first base in the singlestranded portion of the nucleic acid sample, the polymerase catalyzesits incorporation into the DNA strand. Each incorporation event isaccompanied by release of pyrophosphate (PPi) in a quantitycorresponding to the quantity of incorporated nucleotide. Incorporationevents can thus be determined by measuring the PPi released. Addition ofdNTPs is typically performed one at a time, each in a separate buffer.Nucleotide incorporation proceeds sequentially along each immobilizedtemplate as each nucleotide is made available in a preselected orprogrammed order.

A variety of native and modified polymerases may be used. Modifiedpolymerases include, for example, native sequences with additions,insertions or replacements, that result in a polymerase that retains thecapacity to facilitate incorporation of a nucleotide into a primedsample. The polymerase may be an exonuclease deficient polymerase. Thelarge or Klenow fragment of DNA polymerase may also be used.

A dATP or ddATP analogue may be selected which does not interfere in theenzymatic PPi detection reaction but which nonetheless may beincorporated by a polymerase into a growing DNA chain withoutinterfering with proper base pairing. Examples of suitable analoguesinclude [1-thio]triphosphate (or α-thiotriphosphate) analogues of deoxyor dideoxy ATP, e.g., deoxyadenosine [1-thio]triphospate, ordeoxyadenosine α-thiotriphosphate (dATPαS) as it is also known. Theseand other analogues are described in U.S. Pat. No. 6,210,891, by Nyren,et al., entitled “Method of sequencing DNA,” the entire disclosure ofwhich is incorporated herein by reference for its teaching concerningsuch analogues.

Single stranded binding protein may be included to extend the length ofsequences that may be sequenced by reducing folding of the singlestranded sample. Thus, for example, the invention includes a dropletmicroactuator including a droplet including single stranded bindingprotein. The droplet may be an amplification-ready droplet includingsingle stranded binding protein. The invention includes methods ofconducting droplet operations on a droplet including single strandedbinding protein.

8.2.4 Detection of Nucleotide Incorporation

Determination of whether a specific base has incorporated at the targetsite may involve quantification of PPi released during the incorporationreaction. As each dNTP is added to a growing nucleic acid strand duringa polymerase reaction, a PPi is released. PPi released under theseconditions can be detected enzymatically e.g. by the generation of lightin the luciferase-luciferin reaction (discussed further below). As theprocess continues, the complementary strand is assembled, and thenucleotide sequence is determined from the signal peaks. The system mayproduce a program, as illustrated in FIG. 5.

8.2.4.1 Conversion of PPi to ATP

In some cases, PPi released during an incorporation event may bedetected indirectly. PPi quantification may, for example, beaccomplished by quantifying ATP produced from APS in the presence of anenzymatic catalyst. ATP sulfurylase quantitatively converts PPi to ATPin the presence of adenosine 5′ phosphosulfate (APS). Thus, in oneembodiment, PPi can be converted to ATP, and the quantity of ATP can bemeasured to determine the quantity of dNTP incorporated during thereaction.

8.2.4.2 Quantification of ATP

Once PPi is converted to ATP, the ATP can be quantified to measure theincorporation of dNTP. As illustrated in FIG. 5, ATP drives theluciferase-mediated conversion of luciferin to oxyluciferin thatgenerates visible light in quantities that are proportional to thequantity of ATP. The light produced in the luciferase-catalyzed reactionmay be detected, e.g., by a charge coupled device (CCD) camera,photodiode and/or photomultiplier tube (PMT). Light signals areproportional to the number of nucleotides incorporated. Detected signalcan be translated into a system output corresponding to the resultswhich is viewable by a user.

Luciferin-luciferase reactions to detect the release of PPi have beendescribed. For example, a method for continuous monitoring of PPirelease based on the enzymes ATP sulphurylase and luciferase referred toas Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay(“ELIDA”) has been described by Nyren and Lundin (Anal. Biochem., 151,504-509, 1985, the entire disclosure of which is incorporated herein byreference.) The use of the ELIDA method to detect PPi in a droplet on adroplet microactuator is one aspect of the present invention. The methodmay however be modified, for example by the use of a more thermostableluciferase (Kaliyama et al., 1994, Biosci. Biotech. Biochem., 58,1170-1171, the entire disclosure of which is incorporated herein byreference). Examples of suitable detection enzymes for the PPi detectionreaction are ATP sulphurylase and luciferase.

In certain prior art applications, a nucleotide degrading enzyme, suchas apyrase, is used to degrade unincorporated dNTPs and excess ATP. Whendegradation is complete, another dNTP is added. Since the reaction takesplace in a single solution, waste products continue to build up assequencing proceeds. An important aspect of the present invention isthat it avoids the requirement for a nucleotide degrading step. Thus,while in some aspects of the invention, a nucleotide degrading step maybe included, in other aspects, the systems and methods of the inventionspecifically omit a nucleotide degrading step. Thus, for example, theanalysis may be accomplished in the absence of a substantial amount ofnucleotide degrading enzyme, e.g., in the absence of a substantialamount of apyrase.

Further, the inventors believe that conducting theincorporation/conversion/detection reactions in the absence ofsubstantial build-up of byproducts will produce a more predictableresult. Thus, for example, where single nucleotide stretches are presentin the sample, it is traditionally difficult to distinguish the specificnumber of incorporations as the length of the single nucleotide stretchincreases. The inventors believe that the clean nature of the reactionof the present invention will lead to greater accuracy andreproducibility for longer single nucleotide stretches. Thus, forexample, the inventors believe that the method of the invention canaccurately detect single nucleotide stretches having 6, 7, 8, 9 10, 11,12, 13, 14, or more nucleic acids with 90, 95, 99 or 99.9% accuracy.

8.2.5 Droplet Operation Protocols

It will be appreciated that, in addition to the protocols describedabove, a variety of droplet operation protocols may be utilized in orderto carry out the sequence analyses of the invention. Thus, for example,conversion to ATP can be accomplished in a single reaction along withthe dNTP incorporation reaction, or the reactions can be performedstepwise: (1) incorporation of dNTP to release PPi, followed by (2)conversion of PPi to ATP. Where the incorporation and conversion stepsare performed together, the system may transport a single dropletincluding the required reagents (dNTP, polymerase, ATP sulfurylase, andAPS). Thus, all reagents required to incorporate a dNTP into theimmobilized sample, release PPi, and react the PPi with APS to yield ATPmay be included in a reservoir on the droplet microactuator as a singlesource reagent for each nucleotide. The source reagent for eachnucleotide may be successively brought into contact with the immobilizedsample so that the reactions may take place, yielding ATP in thepresence of a complimentary dNTP.

Where the incorporation and conversion steps are performed separately,the primer, polymerase, dNTP, sulfurylase, and APS may be provided fromseparate sources as separate reagent droplets which are merged togetherto perform the reactions of the invention. Alternatively, some or all ofthese reagents may be provided from a single source as a pyrosequencingreagent droplet which is brought into contact with the immobilizedsample in order to conduct the reactions of the invention. For example,a droplet including the dNTP and polymerase may be brought into contactwith the immobilized sample, so that the base will incorporate ifcomplimentary, thereby releasing PPi. The droplet potentially includingthe PPi may then be transported away from the immobilized sample andcombined with a droplet including the ATP sulfurylase and APS to produceATP. Alternatively, a droplet including the ATP sulfurylase and APS maybe combined with the droplet potentially including the PPi in thepresence of the immobilized sample to produce ATP.

Further, while FIG. 4 illustrates the priming step 4 andincorporation/conversion steps 5 as occurring sequentially, it will beappreciated that they can be separated out further or they can all beincorporated into a single step. In other words, each specific reagentmay be added to the reaction at the appropriate time in one or moredroplets or any combination of multiple reagents may be providedtogether in a single droplet or series of droplets. Thus, in oneembodiment, the droplet microactuator transports one or more dropletsinto contact with the single stranded sample, wherein one or moredroplets together include the following reagents (or their functionalequivalents) provided together or in separate droplets: primer,polymerase, and dNTP in any combination and in any order producing theresult that the primer hybridizes with the sample to form a doublestranded portion, the polymerase catalyzes incorporation of anycomplimentary dNTP into a target site at the first single stranded baseposition adjacent to the double stranded portion, thereby releasing PPi.

In another embodiment, the droplet microactuator transports one or moredroplets into contact with the single stranded sample, and the one ormore droplets together include the following reagents (or theirfunctional equivalents): primer, polymerase, dNTP, sulfurylase, and APS,in any combination and in any order producing the result that the primerhybridizes with the sample to form a double stranded portion, thepolymerase catalyzes incorporation of any complimentary dNTP into atarget site at the first single stranded base position adjacent to thedouble stranded portion, thereby releasing PPi, and the ATP sulfurylaseconverts any PPi to ATP in the presence of APS. Base incorporation isdetermined by quantifying the quantity of PPi released.

The droplet potentially comprising ATP may be merged via dropletmicroactuation techniques with a droplet comprising reagents, such asluciferase and luciferin, for facilitating detection of any ATP.Similarly, a droplet including luciferin and potentially comprising ATPmay be merged via droplet microactuation techniques with a dropletincluding luciferase for detection of any ATP. Further, a dropletincluding luciferase and potentially comprising ATP may be merged viadroplet microactuation techniques with a droplet including luciferin fordetection of any ATP.

Droplets for the detection reaction may be merged in the presence of orapart from the immobilized sample. For example, a luciferase/luciferindroplet may be merged with the droplet potentially including ATP in thepresence of the immobilized sample. Alternatively, the dropletpotentially including ATP may be separated from the immobilized sampleto be merged with a luciferase/luciferin droplet. In either case, themerging of droplets including reagents that produce a light signal maybe accomplished in proximity to the sensor in order to maximize theamount of light detected.

When the droplet potentially including ATP is transported away from theimmobilized sample to be merged with a luciferase droplet, the transportstep may include an incubation step in order to maximize the productionof ATP for detection in the fluorescence reaction. In other words, thisincubation may be accomplished during transport, or the droplet may betemporarily stored for incubation prior to the fluorescence reaction.The droplet microactuator may include an incubation zone for thispurpose. The incubation zone may or may not include a heating element tocontrol temperature in the zone. The incubation zone may or may notinclude a wall separating the zone from the remainder of the dropletmicroactuator. The incubation zone may include an array of electrodes tofacilitate transport of droplets into and out of the zone. The zone isscalable and may include electrodes for transporting and storing tens,hundreds or more droplets within the incubation zone.

In the practice of the invention, one or more detection reagents may bespecifically excluded from the polymerase reaction step so that anysignal will not be emitted during the polymerase reaction. For example,in one embodiment, a detection enzyme is not added to the reaction mixfor the polymerase step. Instead, the droplet used to conduct thepolymerase step is transported away from the immobilized sample, thenmerged with a droplet including the detection enzyme in range of asensor for detecting signal from any resulting reaction.

The reaction mix for the polymerase reaction may thus include at leastone dNTP, polymerase, APS, and ATP sulfurylase, and may optionallyinclude luciferin, while lacking any significant amount of luciferase.In this way, the dNTP incorporation reaction may be separated from thedetection reaction. The detection reaction may thus be conducted in thepresence of the sensor, e.g., as illustrated in FIG. 5, in order tomaximize the detection signal. For example, where the detection signalincludes light, the detection reaction may be completed in range of asensor for detecting light emitted from any resulting light-producingreaction.

Further, a detection reaction and a subsequent incorporation reactionmay be conducted in parallel, thereby expediting sequencing speed.Similarly, an incorporation reaction may be conducted, the output of aprevious incorporation reaction may be incubated, and the output of aprevious incubation may be subjected to detection all in parallel inseparate droplets, thereby expediting sequencing speed.

Unlike certain processes of the prior art, the droplet microactuatorapproach of the present invention avoids mass transport effects.Reagents may be brought directly into contact with each sample, withoutrequiring a flow of reagents across multiple samples. Where magneticallyresponsive beads are used, they may be maintained in place using amagnetic field and/or they may be transported from place-to-place indroplets. Reagents may be transported from a reagent source directly toa sample without coming into contact with other samples and potentiallycausing cross-contamination. Diffusion of byproducts is avoided byisolation of droplets in the filler fluid. Wells packed with beads,e.g., stabilizing beads, which may interfere with light detection mayalso be avoided. A wash containing apyrase may be used betweenapplications of nucleotides, but its use is not necessary in thepractice of the invention, and in some embodiments, it is specificallyavoided.

Between applications of each new set of reagents to the immobilizedsample, it may be washed, e.g., with a buffer solution. Various surfacewashing protocols are described in Section 8.6.

8.2.6 Applications

The nucleic acid amplification and sequencing methods, devices andsystems of the invention are useful in a wide variety of settings, suchas scientific research, medical and veterinary diagnostics,phramacogenomics, genomic sequencing, gene expression profiling,detection of sequence variation, forensics, and environmental testing.Due to the portable size enabled by the droplet microactuator of theinvention, sequencing for the applications described herein can, ifdesirable, be accomplished at the point-of-sample collection.

In one embodiment, the invention may provide an influenza test panel. Inthis embodiment, the system may accept a biological sample as input,process the sample to prepare target influenza virus nucleic acids foramplification, conduct amplification using the protocols of theinvention, and detect the presence of target influenza nucleic acids.The biological sample may, for example, be collected from anasopharyngeal swab.

In another embodiment, the invention may provide a respiratory infectionpanel. In this embodiment, the system may accept a biological sample asinput, process the sample to prepare nucleic acids from commonrespiratory pathogens, such as bacteria, viruses and/or fungi, foramplification, conduct amplification using the protocols of theinvention, and detect the presence of target respiratory pathogennucleic acids. In an extended version of the respiratory infectionpanel, the panel may include testing for atypical infections such asthose affecting immuno-compromised patients. The biological sample may,for example, be provided by or obtained from a nasopharyngeal swab.Examples of respiratory pathogens suitable for detection using arespiratory infection panel of the invention include S. pneumoniae, H.influenzae, Legionella, Chlamydia, Mycoplasma; viruses such asinfluenza, RSV, coronavirus, parainfluenzae, adenovirus,metapneumovirus, bocavirus, hantavirus; and fungi such as Pneumocystis,Aspergillus, Cryptococcus.

For sequencing lengthy nucleic acids, e.g., whole genomes, samples maybe broken into smaller overlapping fragments (e.g., 100-1000 bp, 200-900bp or 300-800 bp), e.g., by digestion with restriction enzymes. Thesmaller fragments may be analyzed using the systems and methods of theinvention. Results may be assembled and edited to reconstruct the longersequence, e.g., by identifying and matching overlaps in the sequencedfragments. Analysis of the fragments may proceed in a parallel manner,in order to expedite the sequencing. Each template may be sequencedmultiple times to enhance accuracy. In this way, entire chromosomes oreven entire genomes may be accurately sequenced.

Genes transcribed in a given set of tissues can be determined from mRNAextracted from cells or tissue. mRNA may be copied into DNA (cDNA) usingreverse transcriptase. The resulting cDNAs may be cloned, and the cloneends from a cDNA library may be sequenced according to the methods ofthe invention to generate EST, which provide an expression profile forthe tissue from which the mRNA was extracted. RNA patterns may in somecases be correlated with disease states and may be sequenced as adiagnostic tool. RNA viruses may also be sequenced.

Once a reference sequence has been obtained for a region of interest(e.g., a gene believed to be involved with a disease), variations of thesequence as found in different individuals or closely related speciescan be identified by selectively resequencing a small portion of knownsequence. Variations may, for example, occur as SNPs; size differences(insertions/deletions); copy number differences (duplications) andrearrangements (inversions, translocations).

Populations of organisms can be sequenced, e.g., from water, soil and/oratmospheric samples. For example, most current knowledge of microbiologystill is derived from individual species that either cause disease orgrow readily as monocultures under laboratory conditions and are thuseasy to study. Sequencing can be used to study the organization,membership, functioning, and relationship of such organisms. Qualitativeanalysis of sequence and gene diversity can thus be obtained fromorganisms that cannot be cultured using conventional techniques.

The systems and methods of the invention may also be used to providegenomically specific diagnostics and treatment. For example, the systemsmay be used to identify genotypic traits that are associated with moreor less favorable treatment outcomes. Results may be used to guidetreatment decisions. Similarly, the systems and methods of the inventioncan be used to identify identifying mutations in infectious organisms orgenetically damaged or altered cells, such as cancers and otherneoplasms, and this information can be used to guide or confirmtreatment decisions. Infectious organisms may, for example, includeviruses, bacteria, parasites or fungi. The invention provides, forexample, a system and method for quick, inexpensive detection of drugresistant strains of bacterial or viruses (e.g., new strains ofdrug-resistant HIV) which is a critical component of combating thesedisease causing organisms.

The systems and methods of the invention may be employed for genetictesting, e.g., to identify DNA segments in a subject that play a role ina specific disease or DNA segments which may be predictive of a specificdisease. For example, linkage may be demonstrated when, within families,one form of the marker is found in those with the disease more oftenthan in blood relatives in whom the disease is absent. Such methods haveproved successful in Huntington disease, cystic fibrosis, breast cancer,and other disorders. Thus, for example, the systems and methods of theinvention may be used to identify mutations in a gene that are onlypresent (in gene dosage sufficient to cause disease) in subjects withdisease or subjects predisposed to develop the disease. In anotherembodiment, the systems and methods of the invention are used toidentify genetic variations for which (1) there is a statisticallysignificant probability that the sequence will be present in people withthe disease, and (2) there is a statistically significant probabilitythat the sequence will not be present in people without the disease.Similarly, the systems and methods of the invention are used to identifygenetic variations for which there is a statistically significantprobability that people with positive test results will get the diseaseand that people with negative results will not get the disease. Further,the systems and methods of the invention may be used to sequence asegment of DNA to identify one or more SNPs.

The systems and methods of the invention may be used in a clinical trialsetting. For example, nucleic acids from persons participating in atrial may be sequenced, and adverse events may be compared with geneticvariation in the trial group to identify a subset of participants withincreased susceptibility to one or more adverse events. Depending on theseverity of the particular adverse event in question, subjects with theassociated genetic variation may, for example, be watched more closely,receive further protective treatment, and/or removed from the trialaltogether. Similarly, efficacy may be compared with genetic variationin the trial group to identify a subset of participants with increasedlikelihood to positive treatment outcomes. Target populations can bedefined based on positive outcomes and/or lack of unduly adverse events.Products can be labeled accordingly. Physicians can test their patientsfor the associated genetic variation and can prescribe products only tothe population subset for which treatment is pharmaceuticallyacceptable.

The invention is also useful for forensic evaluations, such as:identifying potential suspects whose DNA may match evidence left atcrime scenes; exonerating persons wrongly accused of crimes; identifyingcrime and catastrophe victims; establishing paternity and other familyrelationships; identifying endangered and protected species as an aid towildlife officials; detecting bacteria and other organisms that maypollute air, water, soil, and food; matching organ donors withrecipients in transplant programs; determining pedigree for seed orlivestock breeds; and authenticating consumables such as caviar andwine; identifying genetically modified foods.

Other examples of applications include: testing for associations betweengenetic variations and subject outcomes, e.g., efficacy, side effectprofile, pharmacokinetics, and/or pharmacodynamics, in the drugdiscovery process; analyzing a subject's genetic profile todifferentiate between potential drug therapies based on genotypicvariation; screening for predisposition for disease so that a subjectcan take steps to monitor, treat, avoid or lessen the severity of agenetic disease; screening to decrease the number of adverse drugreactions in a patient population; screening to enable the use of a drugwhich is not safe in a genetically identifiable population subset; andmonitoring of gene therapies.

8.3 Affinity-Based Assays

The invention provides methods, devices and systems for conductingdroplet-based, affinity-based assays, such as affinity-based assays.These assays include any assay in which a compound having a bindingaffinity for an analyte is contacted with the analyte or a samplepotentially including the analyte using droplet operations. For example,the compound having a binding affinity for an analyte may be provided ina droplet and transported into contact with an analyte which is presentin another droplet on a droplet microactuator or is immobilized on asurface of a droplet microactuator. As another example, the compoundhaving binding affinity for the analyte may itself be immobilized on thesurface of a droplet microactuator and/or on the surface of beadsincluded on a droplet microactuator, and a droplet including the analyteor potentially including the analyte may be brought into contact withthe immobilized antibody.

It will be appreciated that a wide variety of affinity-based assayprotocols are possible within the scope of the invention. Examples ofaffinity-based assay formats include direct affinity-based assays,indirect affinity-based assays, and competitive affinity-based assays.The assays may be employed to detect the presence of a target analyte,and may also in some cases be used to quantify the target analytepresent in a sample. In a competitive assay, a droplet including asample antibody and labeled antibody is contacted with a surface. Thesample antibody competes with labeled antibody for binding to antigenadsorbed onto the surface. A variant of this approach involves linkingthe antigen to the surface via an intermediary linker. For example, thelinker may be an antibody. The capture bridge assay uses a dropletincluding a sample antibody to link antigen adsorbed to the surface withantigen in solution. Another approach involves the use of biotinylatedantigen and a streptavidin coated solid phase. Another approach involvesbinding the sample antibody to antigen immobilized on the solid phase.The bound antibody may be detected with isotype specific labeled secondantibody. Excess antibody can be washed off using the droplet protocolsof the invention.

It will be appreciated that an important aspect of the inventioninvolves the ability to conduct droplet operations using each of therequired affinity-based assay reagents and/or samples on a dropletmicroactuator. For example, the invention includes:

-   (1) a droplet microactuator comprising thereon a droplet comprising    any one or more of the reagents and/or samples described herein for    conducting affinity-based assays;-   (2) a device or system of the invention comprising such droplet    microactuator;-   (3) a method of conducting droplet operations on or otherwise    manipulating a droplet making use of such droplet microactuator or    system; and/or-   (4) a method of conducting an droplet-based affinity-based assay    making use of such droplet microactuator or system.

For example, the droplet operations may include one or more of thefollowing: loading a droplet into the droplet microactuator; dispensingone or more droplets from a source droplet; splitting, separating ordividing a droplet into two or more droplets; transporting a dropletfrom one location to another in any direction; merging or combining twoor more droplets into a single droplet; diluting a droplet; mixing adroplet; agitating a droplet; deforming a droplet; retaining a dropletin position; incubating a droplet; heating a droplet; vaporizing adroplet; cooling a droplet; disposing of a droplet; transporting adroplet out of a droplet microactuator; other droplet operationsdescribed herein; and/or any combination of the foregoing. Various othermethods, devices, systems, and other aspects of the invention will beapparent from the ensuing discussion.

8.3.1 Samples and Sample Preparation

The invention provides droplet-based affinity-based assays which areuseful for detection of a wide variety of analytes. For example, anyanalyte that can bind with specificity to an affinity molecule, such asan antibody, is suitable for detection using the systems of theinvention. A single sample may be analyzed for one or more targetanalytes. Analytes may, for example, be biological analytes or syntheticanalytes. Examples include analytes in the following categories:analytes from human sources, analytes from animal sources, analytes fromplant sources, analytes from bacterial sources, analytes from viralsources, and analytes from spore sources. Analytes may, for example,include proteins, peptides, small molecules, and various biomolecules,such as carbohydrates, lipids, and the like. In one embodiment, samplesare contacted with immobilized antibody (e.g., antibody immobilized onbeads), prior to introduction of the immobilized antibody onto thedroplet microactuator.

An illustrative droplet microactuator 600 suitable for conductingimmunoassays is illustrated in FIG. 6. This embodiment may employ twosubstrates, a first substrate 601 a and a second substrate 601 b spacedapart in a substantially parallel fashion to provide an interveningspace.

Multiple fluid ports or reservoirs may be provided in the interveningspace, such as wash buffer reservoirs 602, sample reservoir 604, primaryantibody reservoir 606, secondary antibody reservoir 608, andimmobilized antibody (e.g., antibody immobilized on beads) reservoir610. Waste areas 612 may also be provided, as well as a magnet 614positioned in a manner which permits interaction between the magnet'smagnetic field and magnetically responsive components located in theintervening space. In this particular embodiment, transport electrodes616 are provided on the first substrate.

8.3.2 Sandwich Affinity-Based Assay

In one embodiment, the invention provides a droplet-based sandwichaffinity-based assay performed on a droplet microactuator. It will beappreciated that a wide variety of protocols are possible within thescope of the invention for conducting sandwich affinity-based assays.

The following droplet-based protocol, which is based on the FIG. 7illustration, is provided as one example only and is not intended to belimiting of the scope of the invention:

-   (1) immobilizing on a surface an antibody (primary) with specificity    for a target analyte;-   (2) washing the immobilized antibody, e.g., using a droplet-based    washing protocol, to remove excess antibody;-   (3) using droplet operations to expose the immobilized antibody to a    sample droplet potentially including the target analyte with binding    affinity for the immobilized antibody;-   (4) washing the immobilized antibody-target analyte complex, e.g.,    using a droplet-based washing protocol, to remove unbound components    of the sample droplet;-   (5) exposing the immobilized antibody (now potentially including the    target analyte bound thereto) to a droplet including a reporter    (secondary) antibody;-   (6) washing away excess reporter antibody, e.g., using a    droplet-based washing protocol;-   (7) optionally, performing additional steps to provide a measurable    parameter or signal;-   (8) measuring the measurable parameter or signal; and-   (9) providing an output indicative of the signal.

Any one or more of the foregoing steps can be performed using dropletoperations on a droplet microactuator as described herein.

8.3.2.1 Immobilizing Antibody

A primary antibody is immobilized on a surface. The surface may, forexample, be a surface of the droplet microactuator, or the surface ofbeads, such as magnetically responsive beads, non-magneticallyresponsive beads, or particles, such as nanoparticles.

Immobilization of the antibody to the surface can be accomplished on thedroplet microactuator using droplet-based protocols. For example,reagents for immobilizing an antibody to a surface may be introduced tothe droplet microactuator, dispensed as discrete droplets andtransported into contact with the surface for deposition. Where thesurface in question is the surface of one or more beads, the beads maybe introduced to the droplet microactuator, dispensed as bufferdroplets, transported on the droplet microactuator, and merged with oneor more droplets including reagents for immobilizing the droplets on thesurface of the beads.

Alternatively, the antibodies may be immobilized off the dropletmicroactuator. For example, antibodies may be immobilized on beads priorto introduction to the droplet microactuator. A variety ofprotein-coated (e.g., streptavidin) and antibody-coated beads arecommercially available. In another embodiment, antibodies may beimmobilized on a surface of the droplet microactuator, e.g., duringmanufacture of the droplet microactuator.

Further, surfaces may be prepared for immobilization of antibodies. Forexample, a surface may be provided including moieties which have anaffinity for an antibody or a binding moiety coupled to an antibody. Theantibody, optionally including the binding moiety, may be brought intocontact with the surface thereby immobilizing the antibody on thesurface. For example, beads pre-coated with streptavadin may beintroduced to the droplet microactuator. Droplet operations may beconducted with droplets including the streptavadin-coated beads, and maybe employed to bring such bead-containing droplets into contact with oneor more droplets including biotinylated antibody to thereby couple theantibody to the beads. As another example, the droplet microactuator mayinclude a streptavidin coated surface on a droplet microactuation path,such that droplet operations may be employed to bring a dropletincluding biotinylated antibody into contact with the streptavidincoated surface and thereby immobilize the antibody on the surface. In ayet another example, capture antibodies can be selectively immobilizedonto the surface of the droplet microactuator, e.g., by patterning thesurface to enable immobilization of antibodies or by couplingphotoactive species directly to the antibodies or to streptavidin andthen immobilizing the antibody selectively by using a direct-write lightsystem or using an optical mask or using other means to selectivelyexpose light.

A wide variety of techniques are available for binding antibodies tosurfaces. For example, the surface may be activated with Protein G toenable the binding of antibody molecules via their Fc domain, leavingthe variable region available for target capture. Antibody may becovalently bound to latex surfaces by reaction of activated antibodywith 1,3-diaminopropane coupled, polystyrene aldehyde/sulfate latex.Surfactant-free sulfate white polystyrene latex beads may be coated withantibody by incubation with antibody and conjugation buffer (30 mMNa₂CO₃, 70 mM NaHCO₃, pH 9.5). Biotinylated antibody can be captured onstreptavidin coated substrates. Antibodies may be covalently bound to amodified surface of the droplet microactuator such as a silane orthiolated layer. Antibodies may be covalently bound to a modifiedsurface of the droplet microactuator (e.g., during assembly or usingdroplet operations to deposit the antibodies on the surface), such as asurface modified with silane or a thiolated surface.

8.3.2.2 Binding Target Analyte to Immobilized Antibody

A sample droplet is contacted with the immobilized antibody to permitany target analyte present in the sample to bind with the immobilizedantibody. This step may be accomplished using droplet operations totransport a sample droplet into contact with the surface on which theantibody is attached, e.g., a droplet including antibody coated beads ora surface of the droplet microactuator on which the antibody isimmobilized. In an alternative embodiment, the surface is a beadssurface, and the bead is contacted with the sample prior to introductioninto the droplet microactuator. The bead may also be washed prior tointroduction into the droplet microactuator. The antibody binds toanalyte from the sample droplet. The binding process may be expedited byincreasing the speed of mass-transport. A few examples of acceleratingmass transport include transport of the droplets at a high speed toenable thorough mixing of the beads with antibodies and the targetanalyte or to replenish the surface of immobilized antibody with targetanalytes. Other means include agitating the incubated droplet in-placeby electrically manipulating the droplet or by a number of externalmeans such as piezoelectric methods of actuation. In absence of anymeans of mass transport, the binding events occur based on diffusion andit could take longer times thereby prolonging the assay times. Theimmobilized antibody (e.g., the beads or the surface) may in someembodiments be subjected to a washing protocol on the dropletmicroactuator, e.g., as described in Section 8.6, to remove excesssample or other materials.

8.3.2.3 Binding Reporter Antibody to Target Analyte

After washing (e.g., the beads or the surface), a droplet comprising areporter antibody having affinity for a different epitope on the analytemay be brought into contact with the washed immobilized antibodypotentially having the captured analyte. The labeled antibody conjugateincludes an antibody coupled to a reporter molecule, such as aradioactive molecule, an enzyme capable of catalyzing a detectablereaction (e.g., a color change, chemiluminescence, fluorescence,chemiluminescence, or electrochemical), a chemiluminescent molecule, ora fluorescent molecule. Depending on the reporter used, the immobilizedantibody (e.g., beads or other surface) including the analyte andreporter antibody may then subjected to a washing protocol, e.g., asdescribed in Section 8.6, to remove excess reporter antibody.

8.3.2.4 Producing and Detecting Measurable Parameter

Bound reporter antibody may be quantified by detecting a signalfacilitated by the reporter antibody. For example, the signal may beradioactivity, color change, luminescence, fluorescence, luminescence,Raman spectra, light scattering approaches, particle/bead aggregation,surface plasmon resonance, Raman spectroscopic effect and the like. Thedetection may be direct or indirect, by detecting the quantity ofantibody coupled to the analyte or by detecting the quantity of unboundantibody.

For approaches requiring further reaction to produce a signal, e.g.,conversion of a non-fluorescent product to a fluorescent product, adroplet including the additional required reactants can be brought intocontact with antibody-antigen-antibody complex in order to facilitatethe further reaction.

Once the reporter antibody has been permitted to bind to the analyte,excess reporter antibody can be washed away using a washing protocol,and droplet operations can be used to bring a droplet including therequired reporter reactants into contact with the immobilized antibody.In one embodiment, the reporter antibody is labeled with an enzyme(e.g., horseradish peroxidase (HRP) or alkaline phosphatase (ALP))capable of catalyzing a reaction which produces a measurable parameter.For example, HRP can be used to catalyze hydrogen peroxide to generatean electrochemical signal which can be detected by measuring the currentor voltage. Detection of bound antibody can be achieved by afluorescence reaction catalyzed by the HRP using Amplex Red and hydrogenperoxide as substrates. Another example employs an alkaline phosphatasemediated conversion of NBT to violet formazan, which can be detected ina droplet colorimetrically. In another approach, a chemiluminescencesubstrate such as luminol or Ps-atto from Lumigen could be catalyzed byHRP to generate a chemiluminescence signal. Other examples of suitabledetection approaches are discussed elsewhere herein (e.g., see 8.3.5).

In one embodiment, the detection step is performed in a droplet on thedroplet microactuator in the presence of a sensor in order to enhance ormaximize capture of signal from the reaction. In another embodiment, thereaction is performed away from a sensor, and the droplet is transportedusing droplet operations into the presence of a sensor for detectionpurposes.

8.3.2.5 Alternative Sandwich Assay Approaches

It will be appreciated that a variety of alternative approaches arepossible. For example the steps need not be performed in the orderdescribed above, e.g., the reporter antibody may be bound to the analyteprior to or at the same time the analyte is exposed to the immobilizedantibody. In another approach, binding of capture antibody, analyte, andreporter antibody can all be performed simultaneously and then presentedto a capture site and then washed. In some approaches, such as surfaceenhanced resonance Raman scattering, washing may not be required.

8.3.3 Competitive Affinity-Based Assay

In one embodiment, the invention provides a competitive affinity-basedassay performed on a droplet microactuator. Analytes for detection bycompetitive affinity-based assay are typically those that are too smallfor binding two antibodies as required by a sandwich assay. It will beappreciated that a wide variety of protocols are possible within thescope of the invention for conducting competitive affinity-based assays.The following droplet-based protocol, which is based on the FIG. 8illustration, is provided for illustration only and is not intended tobe limiting of the scope of the invention:

-   (1) immobilizing on a surface an antibody with specificity for a    target analyte;-   (2) washing the immobilized antibody, e.g., using a droplet-based    washing protocol, to remove excess antibody;-   (3) providing a sample droplet potentially including target analyte    and including a reporter analyte;-   (4) exposing the immobilized antibody to the combined target    analyte/reporter analyte droplet so that the reporter analyte    competes with any target analyte for binding sites;-   (5) washing the substrate to remove unbound analyte and reporter    analyte;-   (6) optionally, performing additional steps to yield a measurable    parameter or signal; and-   (7) quantifying bound reporter analyte, wherein the quantity of    reporter analyte is inversely proportional to the quantity of target    analyte.

Any one or more of the foregoing steps can be performed using dropletmanipulation techniques on a droplet microactuator as described herein.Each of the steps is discussed in further detail in the ensuingsections.

8.3.3.1 Immobilizing Antibody

The antibody may be immobilized as described above in Section 8.4.1.1.

8.3.3.2 Competitive Binding

A droplet including the sample potentially including a target analyte iscombined with a droplet including the reporter analyte, and the combineddroplet is brought into contact with the immobilized antibody.Alternatively, the mixture of target and reporter is accomplished duringloading of the droplet microactuator.

The reporter analyte may be made by coupling the reporter nucleic acidto the analyte using any of a variety of conjugation methods. In oneembodiment, the analyte portion is modified with a molecule, such asbiotin, which generates a secondary capture site for immobilizing astreptavidin sensor DNA complex. The coupling of biotin to the analytemust not unduly interfere with its binding to the capture antibody. Thebiotinylated material may in some cases compete equally with the analytefrom the test sample. Coupling of the biotinylated analyte to a reportermolecule can occur before or after the biotinylated analyte is capturedby the immobilized antibody.

For example, in one embodiment, the assay is performed by mixing adroplet with a known quantity of biotinylated analyte with the sampledroplet potentially containing an unknown quantity of unmodifiedanalyte. A droplet including the biotinylated analyte is combined with adroplet potentially including the target analyte. The combined dropletis contacted with the immobilized antibody so that the biotinylatedanalyte and the target analyte (if any) compete for binding to theimmobilized antibody. The quantity of biotinylated analyte bound isinversely proportional to the quantity of analyte in the test droplet.The immobilized antibody (e.g., the beads or the surface) may then besubjected to a washing protocol, e.g., as described in Section 8.6, toremove excess reporter analyte.

8.3.3.3 Detecting the Reporter Analyte

After washing, a droplet with a streptavidin-biotin-reporter complex isadded to a droplet including the washed beads or otherwise brought intocontact with a surface including the immobilized antibody. Thestreptavidin-biotin-reporter complex binds to any biotinylated analytethat was captured by the antibody on the bead.

8.3.3.4 Alternative Competitive Assay Approaches

The competitive affinity-based assay described here is only one exampleof a droplet microactuator protocol suitable for execution on thedroplet microactuator of the invention. A variety of alternatives arepossible within the scope of the invention. For example, the steps arenot limited to the order given. The antibody can be bound to the targetanalyte/reporter analyte before it is immobilized by combining a dropletincluding the free antibody with one or more droplets including thetarget analyte/reporter analyte, after which the antibody may be broughtinto contact with the surface for immobilization. The reporter analytemay be conjugated with the reporter nucleic acid on the dropletmicroactuator by combining droplets including the two reagents. Adroplet including the reporter analyte may be combined with a dropletincluding the reporter nucleic acid to affect conjugation before orafter the reporter analyte is exposed to the captured antibody.

In another embodiment, a competitive assay is performed by mixing adroplet with a known amount of enzyme-labeled analyte with a sampledroplet containing the target analyte which is then further mixed with adroplet containing antibodies. Competition ensues for binding sitesbetween the labeled and target analytes. The activity of the enzyme isreduced upon binding of the enzyme-labeled analyte with the antibody andthis can be monitored through a number of different types oftransduction events, e.g., absorbance, in order quantify theconcentration of the target analyte in the sample. For example, adroplet containing Vancomycin labeled with glucose-6-phosphatedehydrogenase (G6P-DH) can be mixed with a sample droplet containingunlabelled Vancomycin which can be further mixed with a dropletcontaining antibodies reactive to Vancomycin, glucose-6-phosphate, andnicotinamide adenine dinucleotide (NAD). The activity of G6P-DH isreduced upon binding to an antibody. G6P-DH converts NAD to NADH,resulting in an absorbance change that is measuredspectrophotometrically at 340 nm. Once a calibration has been performedwithin the droplet microactuator, the Vancomycin concentration in eachunknown sample can be determined using the stored calibration curve andthe measured absorbance obtained in the assay of the sample. Otheranalytes that can be detected using the same method includes Valproicacid, Tobramycin, Gentamicin, and Caffeine.

8.3.4 Other Affinity-Based Assay Protocols

The competitive affinity-based assay described here is only one exampleof a droplet microactuator protocol suitable for execution on thedroplet microactuator of the invention. A variety of alternatives arepossible within the scope of the invention. For example, the dropletmicroactuator system of the invention enables multiple affinity-basedassays to be simultaneously performed on a single sample or a singleaffinity-based assay to be performed on multiple samples or acombination thereof. Further, affinity-based assays may be performedalong with other tests, such as PCR and/or immuno-PCR.

A variety of alternative assay types may be executed using droplet-basedprotocols in light of the instant specification. Examples includeimmunoprecipitation assays; immunoradiometric assays; heterogeneousenzyme labeled affinity-based assays in which the quantitation of theantibody bound and unbound fractions requires a physical separation ofthese two fractions; homogeneous (non-separation) enzyme labeledaffinity-based assays which do not require a physical separation ofthese two fractions because the unbound and antibody bound fractions canbe distinguished functionally. For immunoprecipitation assays, dropletsincluding reagents for conducting the immunoprecipitation assays arecombined on a droplet microactuator to conduct the immunoprecipitationassay. Immunoprecipitation may be detected using a light scatteringdetector.

While most of the approaches discussed thus far involve immobilizationof the antibody or the analyte, immobilization is not required in alldroplet-based immunoassays of the invention. For example, the inventionincludes a homogenous droplet-based enzyme-multiplied immunoassay inwhich the labeled antibody includes an enzyme that is inactivated whenbound to the primary antibody. Enzymatic activity is approximatelyproportionate to the analyte concentration. The approach generallyincludes combining on a droplet microactuator droplets for conductingthe droplet-based enzyme-multiplied immunoassay and measuring theresulting enzymatic activity.

In another method, the light scattering properties of theantigen/antibody complex will be altered upon a binding event, and thischange can be monitored by detecting light scattering changes in thereaction droplet on the droplet microactuator, such as turbiditymeasurements, to identify and/or quantitate the capture events. Forexample, a physiological sample droplet on the droplet microactuatorcontaining immunoglobulins such as IgA, IgG, and IgM (after samplepreparation including dilution and addition of polymers) orapolipoproteins such as ApoA1, ApoB (after sample preparation includingdilution and addition of polymers or surfactants) can be combined usingdroplet operations with a droplet containing respective antibodies that,upon occurrence of binding events, results in a change in the turbidityof the combined droplet which can be monitored spectrophotometrically. Afew examples of other analytes that can be measured using this techniqueinclude α₁-antitrypsin (AAT), transferrin, prealbumin, haptoglobin,complement C3, and complement C4.

Another class of immunoassays suitable for use in the present inventioninclude agglutination assays which can also be performed in the dropletformat. A droplet containing the analyte is mixed with a dropletcontaining particles, for example latex particles, with the captureantibody or antigen bound to the particles. If the target analyte ispresent in the sample, the latex particles start to agglutinate togetherand it can be quantified by measuring the absorbance.

The system provides multiplexed affinity-based assays. In oneembodiment, the system has the ability to detect 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50 or more analytes using immunoassays. In some highthroughput settings, the system provides for multiplexed detection of96, 384, 1536, or higher number of analyses either in serial fashion onin parallel fashion. The analytes may, for example, include analytesfrom natural or non-natural sources. In another embodiment, theinvention has the ability to execute affinity-based assay protocols fordetecting one or more analytes from any 2, 3, 4 or 5 the followingcategories: analytes from bacterial sources, analytes from viralsources, analytes from fungal sources, protein toxin analytes, and smallmolecule toxin analytes.

The system may be programmed to repeat assays as needed to increaseconfidence for a single target result. Importantly, the dropletmicroactuator system can be programmed to effect confirmatory re-testingof positives to reduce the possibility of false positives. The systemmay also be programmed and configured to permit storage of testedsamples on the droplet microactuator for subsequent additionallaboratory testing.

An important advantage of the invention is the capability of the dropletmicroactuator to quickly and accurately produce calibration curves. Thedroplet microactuator can accurately and reproducibly dispense dropletsof a solution of known concentration of control analyte and can dilutesuch droplets by combining them with buffer droplets to provide a seriesof control droplets having varied concentrations of control analyte.These control droplets can be taken through the same protocol as sampledroplets to produce a calibration curve. The calibration curve can beused to determine quantities of analyte in sample droplets.

8.3.5 Other Detection Approaches for Affinity-Based Assays

A wide variety of detecting approaches are available for use in thedroplet-based affinity-based assays of the invention. The selectedapproach will be capable of directly or indirectly producing a signal ina droplet-based affinity-based assay. The signal may be detectable by asensor positioned in contact with or in close proximity with thedroplet. Examples of signals suitable for use in the affinity-basedassays include signals produced from radioisotopic labels, fluorescentlabels, luminescent labels, electroluminescent labels microparticles,nanoparticles, enzymatic reactions, aggregation compounds, Raman-activedyes, electroactive labels, and labels affecting conductivity. Examplesof suitable radioisotopic labels include ⁵⁷Co, ³H, ³⁵P, ³⁵S, and ¹²⁵I.In one embodiment, radioisotopic labels are used in a scintillationproximity assay (SPA) on a droplet microactuator. SPA's enable detectionof binding events without requiring a washing step. The radiolabel may,for example, be incorporated into a competitive analyte in a competitionassay or in a secondary antibody in a sandwich assay.

Radiolabels that emit alpha or weak beta particles are preferred. TheSPA is conducted in proximity to a fluorophore that emits light uponexposure to a radiolabel. For example, the fluorophore may be providedin a bead, in a surface of the droplet microactuator to which an antigenor primary antibody is bound, or in nanoparticle coupled to an antigenor primary antibody. Examples of suitable luminescent labels includeacridinium ester, rhodamine, dioxetanes, acridiniums, phenanthridiniumsand various isoluminol derivatives. Examples of suitable fluorescentlabels include fluorescein and Eu³⁺. Examples of suitable enzymaticlabels include those which produce visible, colored, fluorescent and/orluminescent products from suitable substrates. For example, suitableenzymes may include penicillinase, horseradish peroxidase,β-galactosidase, urease, deaminases and alkaline phosphatase. Examplesof suitable nanoparticles include metal nanoparticles. Furtherinformation about detection approaches suitable for affinity-basedassays of the invention is provided in Section 8.11.

8.3.6 Sample Size and Assay Speed

Implementation on a digital microfluidic platform will dramaticallyreduce the equipment size and cost, primarily by miniaturizing allliquid handling functions. Assays can, in some embodiments, be performedon less than 100^(th) or 1000^(th) of the sample and reagent volumescurrently used with equal sensitivity and specificity. In oneembodiment, the system will typically perform affinity-based assaysusing samples at droplet volumes of 1 μL or less, or 100 nanoliters orless.

Other advantages include reduced time to results due to faster kineticsin the miniaturized format for the assays and higher throughput due tomultiplexing. For example, in one embodiment, the system executesaffinity-based assay protocols for detecting 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50 or more analytes in less than about 60, 50, 40, 30, 20,15, 10, 5 or 2 minutes.

8.3.7 Applications of Droplet-Based, Affinity-Based Assays

As noted above, the affinity-based assays of the invention are usefulfor detecting a wide variety of molecules. Any molecule which binds withaffinity to an affinity molecule, such as an antibody, is a suitableanalyte. Analytes may, for example, include biological molecules orsynthetic molecules. Biological molecules may, for example, includemolecules from plants, animals, microbes, and viruses. Syntheticmolecules may, for example, include industrial byproducts, pollutants,and pharmaceuticals. Analytes may include toxins or analytes indicativeof the presence of specific biological organisms, e.g., infectiousdiseases, or toxins that are employed in bioterrorism or biologicalwarfare. Examples of such organisms include anthrax, avian influenza,botulism, hantavirus, legionnaires' disease, pneumonic plague, smallpox,tularemia, and viral hemorrhagic fevers (VHFs). Other examples includeanalytes associated with monitoring the immunogenicity of vaccinestargeting cancer, chronic infectious diseases (e.g., HIV, malaria,hepatitis C, candidemia), immunology (e.g., allergy, autoimmunity),endocrinology (thyroid, non-thyroid), drug testing (e.g., drugs ofabuse, therapeutic drugs) and detection of bioterrorism agents (e.g.,anthrax, smallpox).

In one embodiment, immunoassay technique is used to test a sample forthe presence of a biological organism, such as a bacteria or a virus. Insome embodiments, the system can achieve extremely sensitive detectionseven down to a single cell. Further, some embodiments may include PCRbacterial typing or variance.

The affinity-based assays of the invention are also useful for detectingchemical, biological or explosive threats. For example, antibody toexplosives can be used to detect the presence of trace amounts ofexplosive in a sample. Thus, the invention also provides a method ofscreening an area for chemical or biological analytes indicative of thepresence of biological, chemical, or explosive weapons.

In one embodiment, the invention provides the capability of detectingmultiple analytes in a single droplet. One way to achieve this resultaccording to the invention involves the use of different antibodies fordifferent analytes at spatially separated locations on the dropletmicroactuator. For example, the droplet microactuator may includemultiple surfaces, each comprising a specific antibody. A single sampledroplet may be manipulated to come into contact with these antibodiesall at once, or sequentially, as the droplet is transported across theantibody regions. Affinity-based assay protocols of the invention may beemployed to detect the presence of analyte bound to the antibody in anyof the various regions. Similarly, multiple analytes can be detected ina single droplet by using different labels to simultaneously detectdifferent analytes in the same spatial area. In another embodiment, thedroplet microactuator includes spatially separated beads, each bead orset of beads having a unique antibody or set of antibodies. Sampledroplets and/or bead containing droplets may be manipulated usingdroplet operations in order to contact a sample droplet with each of thebeads or sets of beads.

8.4 Immuno PCR

The invention provides a droplet-based immuno PCR (pick) for sensitivelydetecting analytes that are available only at trace levels. Thisinvention combines the various means of affinity-based assays using adetector antibody on a droplet-based platform and utilizes a nucleicacid strand as a label. This nucleic acid strand is amplified usingamplification techniques (e.g., see Section 8.1).

It will be appreciated that an important aspect of the inventioninvolves the ability to conduct droplet operations using each of therequired iPCR reagents and/or samples on a droplet microactuator. Forexample, the invention includes:

-   (1) a droplet microactuator comprising thereon a droplet comprising    any one or more of the reagents and/or samples described herein for    conducting iPCR protocols;-   (2) a device or system of the invention comprising such droplet    microactuator;-   (3) a method of conducting droplet operations on or otherwise    manipulating a droplet making use of such droplet microactuator or    system; and/or-   (4) a method of conducting an droplet-based affinity-based assay    making use of such droplet microactuator or system.

For example, the droplet operations may include one or more of thefollowing: loading a droplet into the droplet microactuator; dispensingone or more droplets from a source droplet; splitting, separating ordividing a droplet into two or more droplets; transporting a dropletfrom one location to another in any direction; merging or combining twoor more droplets into a single droplet; diluting a droplet; mixing adroplet; agitating a droplet; deforming a droplet; retaining a dropletin position; incubating a droplet; heating a droplet; vaporizing adroplet; cooling a droplet; disposing of a droplet; transporting adroplet out of a droplet microactuator; other droplet operationsdescribed herein; and/or any combination of the foregoing. Various othermethods, devices, systems, and other aspects of the invention will beapparent from the ensuing discussion.

8.4.1 Sandwich iPCR

In one embodiment, the invention provides a droplet-based sandwich iPCRperformed on a droplet microactuator. In general, the sandwich iPCRinvolves:

-   (1) immobilizing on a surface an antibody with specificity for a    target analyte;-   (2) washing the immobilized antibody, e.g., using a droplet-based    washing protocol;-   (3) exposing the immobilized antibody to a sample droplet    potentially including the target analyte;-   (4) washing the immobilized antibody, e.g., using a droplet-based    washing protocol;-   (5) exposing the immobilized antibody (now potentially including the    target analyte bound thereto) to a droplet including a second    antibody conjugated to a nucleic acid molecule;-   (6) washing the immobilized antibody, e.g., using a droplet-based    washing protocol;-   (7) amplifying the nucleic acid and detecting the amplification (if    any) to determine the presence and quantity of captured target    analyte.

Any one or more of the foregoing steps can be performed using dropletmanipulation techniques on a droplet microactuator as described herein.Each of the steps is discussed in further detail in the ensuingsections.

8.4.1.1 Immobilizing Antibody

A primary antibody is immobilized on a surface. The surface may, forexample be a surface of the droplet microactuator, or the surface ofbeads, such as magnetically responsive beads. Immobilization of theantibody to the surface can be accomplished on the droplet microactuatorusing droplet-based protocols. For example, reagents for immobilizing anantibody to a surface may be introduced to the droplet microactuator,dispensed as discrete droplets and transported into contact with thesurface for deposition. Where the surface in question is the surface ofone or more beads, the beads may be introduced to the dropletmicroactuator, dispensed as droplets in a buffer, transported, andmerged with one or more droplets including reagents for immobilizing thedroplets on the surface of the beads.

A wide variety of techniques are available for binding antibody tosurfaces. For example, the surface may be activated with Protein G toenable the binding of antibody molecules via their Fc domain, leavingthe variable region available for target capture. Antibody may becovalently bound to latex surfaces by reaction of activated antibodywith 1,3-diaminopropane coupled, polystyrene aldehyde/sulfate latex.Surfactant-free sulfate white polystyrene latex beads may be coated withantibody by incubation with antibody and conjugation buffer (30 mMNa₂CO₃, 70 mM NaHCO₃, pH 9.5). Biotinylated antibody can be captured onstreptavidin coated substrates. Light-directed immobilization can alsobe performed, e.g., as described elsewhere in the present disclosure.

8.4.1.2 Binding Target Analyte to Immobilized Antibody

A sample droplet is contacted with the immobilized antibody to permitany target analyte present in the sample to bind with the immobilizedantibody. This step may be accomplished by transporting a sample dropletinto contact with a surface of the droplet microactuator on which theantibody is immobilized. In another embodiment, the step may beaccomplished by merging a sample droplet with a droplet including beadson which the antibody has been immobilized. The antibody binds to theanalyte from the sample droplet. The immobilized antibody (e.g., thebeads or the surface) may be subjected to a washing protocol, e.g., asdescribed in Section 8.6.

8.4.1.3 Binding Antibody-NA to Target Analyte

After washing (e.g., the beads or the surface), a droplet comprising anantibody-NA conjugate having affinity for a different epitope on theanalyte may be brought into contact with the washed immobilized antibodypotentially having the captured analyte. The antibody-NA conjugateincludes a nucleic acid molecule coupled to the antibody. The nucleicacid molecule serves as the nucleic acid template for amplification. Theimmobilized antibody (e.g., the beads or the surface) are then subjectedto a washing protocol, e.g., as described in Section 8.6.

8.4.1.4 Amplifying the Nucleic Acid

The nucleic acid is amplified, e.g., as described in Section 8.1. Thequantity of amplified product produced is measured, e.g., using realtime fluorescence detection, electrochemical and/orelectrochemiluminescent detection. The quantity of PCR product producedcorrelates with the quantity of bound antibody-DNA, which depends inturn on the quantity of analyte present in the sample droplet.

8.4.1.5 Alternative Approach

In an alternative embodiment, the order of these steps is generallyreversed to perform nucleic acid amplification followed by anaffinity-based assay that results in an optical or electrical signal. Inthis sequence, an immunoassay would be performed to monitor nucleic acidamplification.

8.4.2 Competitive iPCR

In one embodiment, the invention provides a competitive iPCR performedon a droplet microactuator. Analytes for detection by competitive iPCRare typically those that are too small for binding two antibodies asrequired by a sandwich assay. In general, the competitive iPCR involves:

-   (1) immobilizing on a surface an antibody with specificity for a    target analyte;-   (2) combining a sample droplet potentially including target analyte    with a droplet including a reporter analyte;-   (3) exposing the immobilized antibody to the combined target    analyte/reporter analyte droplet so that the reporter analyte    competes with any target analyte for binding sites;-   (4) washing the substrate to remove unbound analyte;-   (5) coupling the bound reporter analyte to a reporter nucleic acid;    and-   (6) amplifying the reporter nucleic acid and monitoring the progress    of the amplification to determine the quantity of unbound reporter    analyte, which for the bound reporter analyte is inversely    proportional to the quantity of target analyte in the sample.

Any one or more of the foregoing steps can be performed using dropletmanipulation techniques on a droplet microactuator as described herein.Each of the steps is discussed in further detail in the ensuingsections.

8.4.2.1 Immobilizing Antibody

The antibody may be immobilized as described above in Section 8.4.1.1.

8.4.2.2 Competitive Binding

A droplet including the sample potentially including a target analyte iscombined with a droplet including the reporter analyte, and the combineddroplet is brought into contact with the immobilized antibody.

The reporter analyte may be made by coupling the reporter nucleic acidto the analyte using any of a variety of conjugation methods. In oneembodiment, the analyte portion is modified with a molecule, such asbiotin, which generates a secondary capture site for immobilizing astreptavidin-DNA complex. The coupling of biotin to the analyte must notunduly interfere with its binding to the primary capture antibody. Thebiotinylated material may in some cases compete equally with the analytefrom the sample. Coupling of the biotinylated analyte to the reporternucleic acid can occur before or after the biotinylated analyte iscaptured by the immobilized antibody.

For example, in one embodiment, the assay is performed by mixing adroplet with a known quantity of biotinylated analyte with the sampledroplet potentially containing an unknown quantity of unmodifiedanalyte. A droplet including the biotinylated analyte is combined with adroplet potentially including the target analyte. The combined dropletis contacted with the immobilized antibody so that the biotinylatedanalyte and the target analyte (if any) compete for binding to theimmobilized antibody. The quantity of biotinylated analyte bound isinversely proportional to the quantity of analyte in the sample droplet.

8.4.2.3 Coupling the Nucleic Acid Reporter

After washing, a droplet with a streptavidin-biotin-reporter nucleicacid complex is added to a droplet including the washed beads orsurface. The streptavidin-biotin-reporter nucleic acid complex binds toany biotinylated analyte that was captured by the antibody on the bead.

8.4.2.4 Amplifying the Nucleic Acid

After washing, the quantity of a streptavidin-biotin-reporter nucleicacid complex immobilized is determined by amplification of the reporternucleic acid. The amplification signal is an inverse measure of thequantity of analyte in the original sample. Amplification may proceed asdescribed in Section 8.1. The presence and quantity of amplified productproduced is measured, e.g., using real time fluorescence detection. Thequantity of reporter analyte that was displaced is proportional to thequantity of target analyte in the sample.

8.4.2.5 Alternative Approaches

The steps are not limited to the order given. For example, the antibodycan be bound to the target analyte/reporter analyte before it isimmobilized by combining a droplet including the free antibody with oneor more droplets including the target analyte/reporter analyte, afterwhich the antibody may be brought into contact with the surface forimmobilization. The reporter analyte may be conjugated with the reporternucleic acid on the droplet microactuator by combining dropletsincluding the two reagents. A droplet including the reporter analyte maybe combined with a droplet including the reporter nucleic acid to affectconjugation before or after the reporter analyte is exposed to thecaptured antibody. A variety of alternatives is possible within thescope of the invention.

8.4.3 Samples and Sample Preparation

A wide variety of analytes may be detected using droplet-based iPCRprotocols of the invention. A single sample may be analyzed for one ormore target analytes. Analytes may, for example, be biological analytesor synthetic analytes. For example, in one embodiment, the analytes areselected from following categories: analytes from bacterial sources,analytes from viral sources, analytes from spore sources, protein toxinanalytes, and small molecule toxin analytes. In one embodiment, thetarget analytes include toxins or analytes indicative of the presence ofspecific biological organisms, e.g., infectious diseases, or toxins thatare employed in bioterrorism or biological warfare. Examples of suchorganisms include anthrax, avian influenza, botulism, hantavirus,legionnaires' disease, pneumonic plague, smallpox, tularemia, and viralhemorrhagic fevers (VHFs).

8.4.4 Immuno-PCR Protocols

The system of the invention enables multiple immuno-PCR tests to besimultaneously performed on a single sample. Further, immuno-PCR testsmay be performed along with other tests, such as PCR and/oraffinity-based assays.

The system provides multiplexed detection. In one embodiment, the systemhas the ability to detect 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50or more analytes. The analytes may, for example, include analytes fromnatural or non-natural sources. In another embodiment, the invention hasthe ability to detect one or more analytes from any 2, 3, 4 or 5 thefollowing categories: analytes from bacterial sources, analytes fromviral sources, analytes from spore sources, protein toxin analytes, andsmall molecule toxin analytes.

The system may be programmed to implement additional tests as needed toincrease confidence for a single target result. Importantly, the dropletmicroactuator system can be programmed to effect confirmatory re-testingof positives to reduce the possibility of false positives. The systemmay also be programmed and configured to permit storage of testedsamples on the droplet microactuator for subsequent additionallaboratory testing.

In operation, the system performs analysis and provides resultsextremely quickly. For example, in one embodiment, the system has theability to detect 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or moreanalytes in less than about 60, 50, 40, 30, 20, 15 or 10 minutes. Inanother embodiment, the system has the ability to detect 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more analytes from any 2, 3, 4 or 5 thefollowing categories: analytes from bacterial sources, analytes fromviral sources, analytes from spore sources, protein toxin analytes, andsmall molecule toxin analytes, and all tests are completed in less thanabout 60, 50, 40, 30, 20, 15 or 10 minutes.

8.4.5 Applications

The iPCR assays provided by the invention are useful for detecting awide variety of molecules present in extremely small quantities. Amongother things, the system is useful for surveillance for a chemical,biological or explosive threat. For example, antibody to explosives canbe used to detect the presence of trace amounts of explosive in asample. Thus, the invention also provides a method of screening an areafor chemical or biological analytes indicative of the presence ofbiological, chemical, or explosive weapons.

In one embodiment, iPCR technique is used to test a sample for thepresence of a biological organism, such as a bacteria or a virus. Insome embodiments, the system can achieve even single-organism detection.Further, some embodiments may include PCR bacterial typing or variance.

8.5 Analysis of Biological Fluids

The invention provides methods, devices and systems for analysis ofblood, various components of blood, and other biological fluids.Illustrative designs for a biological fluid analyzer are shown in FIGS.9 and 17.

FIG. 9 illustrates one embodiment of a biological fluid analyzer 900. Inthis embodiment, various modules may be provided for conductingbiological fluid analysis, such as, for example, detection ofmetabolites (e.g., glucose, lactate, blood urea nitrogen, andcreatinine), electrolytes (e.g., K⁺, Cl⁻, and Na⁺), proteins, andenzymes. These various modules may include amperometric module 902,potentiometric module 904, optical module 906, and conductometric module908.

Another embodiment of a biological fluid analyzer 1700 is illustrated inFIG. 17. In this embodiment, multiple fluid ports or reservoirs may beprovided such as antibody reservoirs 1701 (such as for bacteriaantibodies, spore antibodies, bacteria AB-DNA, spore AB-DNA, proteintoxin antibodies, small molecule antibodies, protein AB-DNA, and smallmolecule SB-A DNA), PCR primer reservoirs 1702, and PCR reagentsreservoirs 1703. Sample port 1704 may also be provided, as well assample reservoir 1705, wash solution area 1706, and waste reservoir1707. Other areas that may be provided include hot temperature area1708, cold temperature area 1709, and detector area 1710.

It will be appreciated that an important aspect of the inventioninvolves the ability to conduct droplet operations using each of therequired biological fluid analysis samples and reagents on a dropletmicroactuator. For example, the invention includes:

-   (1) a droplet microactuator comprising thereon a droplet comprising    any one or more of the reagents and/or samples described herein for    conducting such biological fluid analysis;-   (2) a device or system of the invention comprising such droplet    microactuator;-   (3) a method of conducting droplet operations on or otherwise    manipulating a droplet making use of such droplet microactuator or    system; and/or (4) a method of conducting an droplet-based    affinity-based assay making use of such droplet microactuator or    system.

For example, the droplet operations may include one or more of thefollowing: loading a droplet into the droplet microactuator; dispensingone or more droplets from a source droplet; splitting, separating ordividing a droplet into two or more droplets; transporting a dropletfrom one location to another in any direction; merging or combining twoor more droplets into a single droplet; diluting a droplet; mixing adroplet; agitating a droplet; deforming a droplet; retaining a dropletin position; incubating a droplet; heating a droplet; vaporizing adroplet; cooling a droplet; disposing of a droplet; transporting adroplet out of a droplet microactuator; other droplet operationsdescribed herein; and/or any combination of the foregoing. Various othermethods, devices, systems, and other aspects of the invention will beapparent from the ensuing discussion.

8.5.1 Sample and Sample Preparation

Examples of suitable samples for use with the droplet microactuator ofthe invention include whole blood, serum, and plasma, and variouscomponents thereof. Venous, arterial, or capillary blood can be used.Examples of other samples usefully analyzed according to the presentinvention include cerebrospinal fluid (CSF), urine, saliva, sweat,tears, amniotic fluid, pleural fluid, milk, cystic fluid, synovialfluid, stool, and semen.

Serum and/or plasma may be extracted from whole blood on the dropletmicroactuator and/or prior to introduction into the dropletmicroactuator. An example of a loading structure 1000 provided for thispurpose is provided in FIG. 10. In this embodiment, fluid is flowed froma reservoir 1002 through a sealing means 1004 into a loading chamber1006 where it comes into contact with a membrane 1008. Permeate passesinto a permeate flow channel 1010 through which it flows into dropletmicroactuator reservoir 1012, assisted by pressure source 1014 whichapplies pressure via channel 1016.

The small size of the device dramatically reduces the volume of samplerequired for routine testing, which is an important concern in manysettings. For example, typical sample sizes will have a volume which isin one embodiment from about 1 mL to about 100 mL, or about 10 mL toabout 10 mL, or about 1 μL to about 10 μL.

8.5.2 Analytes

The invention provides a versatile droplet microactuator system capableof performing an array of tests on a single sub-microliter droplet ofblood or any physiological sample of about ˜0.5 μL. Examples of suitabletests include metabolites (e.g., glucose, creatinine, lactate, bloodurea nitrogen), electrolytes/elements (e.g., K+, Na+, Cl—, P, Mg, Li,Ca, Fe), gases (e.g., pH, pCO₂, NH₃), enzymes (alkaline phosphatase(ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT),lactate dehydrogenase (LDH), Lipase, Creatine Kinase, Creatine KinaseMB), proteins (albumin, c-reactive protein (CRP), urine microalbumin,urine protein, cerebrospinal fluid protein, serum total protein), andhematocrit. Other analytes include glycated hemoglobin (Alc),hemoglobin, uric acid, triglycerides, cholesterol, high densitylipoprotein (HDL) and low density lipoprotein (LDL).

8.5.2.1 Metabolites

The invention is useful in conducting a variety of enzyme-coupledassays, such as for glucose, blood urea nitrogen, creatinine, andlactate based on electrochemical or optical detection. In someembodiments, glucose, lactate, and creatinine are measured through theamperometric detection of H₂O₂ in an enzyme-coupled assay performed onthe droplet microactuator.

In some embodiments, the invention includes an electrochemistry modulewith electrodes for amperometric and/or potentiometric detection ofmetabolites (e.g., glucose, lactate, blood urea nitrogen, creatinine).

8.5.2.2 Electrolytes

For quantifying various ions (e.g., ammonia, bromide, cadmium, calcium,chloride, copper, cyanide, fluoride, fluorborate, iodide, lead, nitrate,perchlorate, potassium, silver/sulfide, magnesium, iron, lithium,phosphorus, sodium, surfactant, thiocyanate) in a sample, such as aprocessed biological sample, the droplet microactuator may be modifiedto include an ion-selective electrode (ISE). A sample droplet may betransported into contact with the ISE for detection of the desired ion.Further, prior to detection, standard droplets may be brought intocontact with the ISE for the purpose of calibration. In someembodiments, the invention includes an electrochemistry module withelectrodes for amperometric, potentiometric, and/or conductometricdetection of electrolytes (e.g., K⁺, Cl⁻, Na⁺).

ISEs can be included in the droplet microactuator for detectingelectrolytes. ISEs can, for example, be included as components of a topand/or bottom substrate and/or as components exposed to a space betweena top and/or bottom substrate or associated with a single substratedroplet microactuator. They can be integrated with transport electrodes.The ISEs are generally arranged so that droplet operations can beemployed to bring a droplet on the droplet microactuator into contactwith an ISE. Various techniques can be used to make ion selectiveelectrodes on the droplet microactuator. Examples include screenprinting, as well as photolithography, etching, and lift-off.

As a specific non-limiting example, Ag/AgCl can be screen printed toprovide working and reference electrodes with a KCl salt bridge.Examples of suitable ionophores in PVC for the fabrication ofion-selective membranes include methyl monensin for Na+, valinomycin forK+, quaternary ammonium chloride for Cl—, and tridodecyl amine for pH.The ion-selective membranes can be made by micro dispensing and/orspray-coating (e.g., thermal/ultrasonic printing).

Electrolytes can be detected in any biological sample. Specificnon-limiting examples include whole blood, plasma and serum, as well asthe examples provided elsewhere in this disclosure.

8.5.2.3 Gases

For quantifying the presence of gasses (e.g., pCO₂, pO₂) or pH, variousspecialized electrodes may be used. As a non-limiting example, a carbondioxide microprobe may be incorporated into a droplet microactuator ofthe invention for detection and/or quantification of carbon dioxide. Themicroprobe may be arranged so that droplet operations can be employed tobring a droplet on the droplet microactuator into contact with themicroprobe. Further, prior to detection, standard droplets may bebrought into contact with the carbon dioxide microprobe, using dropletoperations, for the purpose of calibration. Corresponding approaches aresuitable for detecting oxygen and/or determining pH. In someembodiments, the droplet microactuator may include electrodes foramperometric, potentiometric, and/or conductometric detection of bloodgases (e.g., pCO₂, pO₂, pH).

As a specific non-limiting example, a Severinghaus-type CO₂ sensor canbe made with the pH electrode made of gold-quinhydrone electrodeimmersed with the internal solid electrolyte made of NaHCO₃, NaCl, and asucrose binder. A gas permeable membrane of polydimethylsiloxane can bedeposited thereon. Digital conditioning electronics (e.g., high inputimpedance amplifiers) can be used to interface with the potentiometricelectrodes.

Gasses can be detected in any droplet on the droplet microactuator.Specific non-limiting examples include droplets including whole blood,plasma, and/or serum, as well as the biological samples describedelsewhere in this disclosure.

8.5.2.4 Enzyme

In some embodiments, the invention includes chemiluminescence assays fordetection of enzymes, such as liver enzymes. Using a series ofdroplet-based multiple enzymatic steps, the ALT and AST assays can bereduced to a final step that produces hydrogen peroxide which can bemeasured quantitatively by absorbance, luminescence, fluorescence, orelectrochemically.

8.5.2.5 Serum Protein

A colorimetric assay may be utilized for detection of total protein in asample. Examples of suitable colorimetric methods include: the Biuretmethod, the Lowry method, the bicinchoninic acid (BCA) assay, andBradford assay. The Biuret method generally involves contacting a sampledroplet with a droplet comprising cupric ions. The cupric ions form acolored complex with proteins. The Lowry reaction approach is based onthe amplification of the biuret reaction by combining with a Folinreagent droplet. A variation of the Lowry assay uses a Bicinchoninicacid (BCA) droplet to permit detection of the cuprous ions generatedfrom cupric ions by reaction with protein in a droplet under alkalineconditions. The Bradford assay approach involves combining the samplewith a droplet comprising Coomassie Blue dye to form a colored complex.In each case, an LED/photodiode setup, e.g., as shown in FIG. 21A, canbe used for monitoring the absorbance. Total protein can be detected inany sample droplet using techniques described herein, including opticalsensing methods based on fluorescence and/or chemiluminescence, as wellas using the affinity-based assay techniques disclosed herein. Specificnon-limiting examples of useful samples for determining total proteinsinclude biological samples, such as whole blood, plasma and serum, aswell as the samples described elsewhere in this disclosure.

8.5.2.6 Hematocrit

Red blood cells can be quantified in a sample droplet using a variety oftechniques. For example, the hemoglobin content can be calculated bymeasuring absorbance at 805 nm. Oxyhemoglobin can then be calculated bymeasuring the absorbance at 650 nm. Results may be obtained by comparingthe absorbance measurements for samples to the absorbance measurementsfor a series of known standards.

In some embodiments, the invention includes an electrochemistry modulewith electrodes for amperometric, potentiometric, and/or conductometricdetection of hematocrit. The droplet microactuator may include aconductometric cell with a pair of electrodes for AC conductometricmeasurement of hematocrit. For a two-substrate droplet microactuator,the electrodes may be located on one or both substrates. The electrodesare arranged so that arranged so that droplet operations can be employedto bring a droplet on the droplet microactuator into contact with theelectrodes.

8.5.3 Multi-Analyte Analyzer

Examples of suitable analytes are glucose, creatinine, lactate, BUN, K⁺,Na⁺, Cl⁻, pH, pCO₂, ALP, total protein, and hematocrit. In oneembodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more analytes areanalyzed on a single droplet microactuator. Preferably, these analytesare selected from glucose, creatinine, lactate, BUN, K⁺, Na⁺, Cl⁻, pH,pCO₂, ALP, total protein, and hematocrit. In one embodiment, glucose,creatinine, lactate, BUN, K⁺, Na⁺, Cl⁺, pH, pCO₂, ALP, total protein,and hematocrit are analyzed on a single droplet microactuator. Otherexamples of suitable analytes include calcium, bilirubin, albumin,clotting time, ALT, and AST.

In some embodiments, assays 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or moreanalytes are processed in parallel on a droplet microactuator system ofthe invention, i.e., one or more processing and/or detecting steps forsuch analyte are accomplished simultaneously with one or more processingand/or detecting steps for another analyte on a single dropletmicroactuator. A droplet microactuator system may execute 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12 or more colorimetric assays for detection of thesame or different analyte types in parallel. The droplet microactuatorsystem may execute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or morechemiluminescence assays for detection of the same or different analytetypes in parallel. The droplet microactuator may execute 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12 or more amperometric assays for detection of thesame or different analyte types in parallel. The droplet microactuatormay execute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more potentiometricassays for detection of the same or different analyte types in parallel.The droplet microactuator may execute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or more fluorescence assays for detection of the same or differentanalyte types in parallel. The droplet microactuator may include 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or more conductometric assays for detectionof the same or different analyte types run in parallel. The dropletmicroactuator will include droplets or reservoirs including reagents forexecuting each of the protocols. The droplet microactuator device and/orsystem will include detection components as needed for executingdetection steps of the protocols.

Moreover, the droplet microactuator may include 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 or more analytes that are processed in parallel, and thesystem may execute 1, 2, 3, 4, 5, 6 or more assay protocols on theseanalytes. The droplet microactuator may include 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 or more analytes that are processed in parallel, and execute1, 2, 3, 4, 5, 6 or more assay protocols on these analytes, where theassays are selected from colorimetric assays, chemiluminescence assays,fluorescence assays, amperometric assays, potentiometric assays andconductometric assays. The droplet microactuator will include dropletsor reservoirs including reagents for executing each of the protocols.The droplet microactuator device and/or system will include detectioncomponents as needed for executing detection steps of the protocols.

Furthermore, various protocols for nucleic acid amplification, nucleicacid sequencing, affinity-based assays, cell handling, bead handling andwashing, and analyte detection protocols may also be readily integratedinto a single droplet microactuator system. In one embodiment, a singledroplet microactuator includes reagents and detection components forconducting nucleic acid amplification and nucleic acid sequencing. Inanother embodiment, a single droplet microactuator includes reagents anddetection components to conduct nucleic acid amplification for detectionof a blood-borne pathogen and reagents and detection components forconducting one or more other assays from those assay types and/or foranalyte types as described herein. In another embodiment, a dropletmicroactuator includes components for manipulating cells along withcomponents and reagents for conducting affinity-based assays.

In short, the invention enables a droplet microactuator system that notonly performs the routine operations of a central lab-based chemistryanalyzer at higher throughput with dramatically lower sample volumes,but also offers better functionality by integrating hematology,pathology, molecular diagnostics, cytology, microbiology, and serologyonto the same platform.

In one embodiment, the invention provides a droplet manipulation moduleintegrated with an optical detection module and an electrochemicaldetection module for analyzing blood gases, electrolytes, enzymes,proteins, and metabolites.

One embodiment of the invention employs a modular design to partitionindependently optimized fabrication processes. For example, all theelectrochemical components can be fabricated on a substrate, all themicrofluidic electrodes can be fabricated on another substrate, and allthe electronics can be fabricated on yet another substrate. A disposablesandwich droplet microactuator can be formed between the electrochemicalmodule and the droplet manipulation module which can be coupled to areusable electronics module for data acquisition and analysis. Opticaldetection modules can be constructed in the analyzer.

8.5.4 Biological Fluid Analysis Detection

The biological fluid analyses described herein make use of a variety ofdetection approaches, e.g., as described in Sections 8.1, 8.2, 8.3, 8.4,8.5, and 8.11.

8.6 Surfaces and Surface Washing Protocols

Various protocols of the invention require surfaces for immobilizationof reactants. For example, surfaces may be used to capture or immobilizetarget components of a droplet, such as cells, other beads,microparticles, nanoparticles, antibodies, proteins, peptides, nucleicacids, small molecules and/or other chemical components. Surfaces usedfor such purposes may, for example, include surfaces of beads,microparticles, nanoparticles, membranes, physical objects, and/ordroplet microactuator surfaces. Various protocols require washing stepin which unbound materials are removed from one or more surfaces.

A sample droplet including one or more target components for capturemay, using droplet operations, be contacted with a surface havingaffinity for such targets. Washing protocols of the invention may beused to remove from the surface unbound components of the sampledroplet. For example, a droplet protocol may be used to bring one ormore droplets including one or more target components into contact withone or more surfaces so that the one or more target components may beimmobilized or captured on the one or more surfaces. A washing protocolmay be executed to remove unbound substances from the one or moresurfaces. Similarly, a droplet protocol may be used to bring one or moredroplets including one or more target components into contact with oneor more beads so that the one or more target components may beimmobilized or captured on the one or more beads. A washing protocol maybe executed to separate unbound substances from the one or more beads.

Washing generally involves bringing one or more washing droplets intocontact with the immobilized surface. Washing may involve agitation ofthe droplets while in contact with the surface. Washing droplets may,for example, include water, deionized water, saline solutions, acidicsolutions, basic solutions, detergent solutions and/or buffers.

Washing protocols of the invention result in highly efficient removal ofunbound substances from the surface. In one embodiment, the inventionprovides method of providing a droplet in contact with a surface with areduced concentration of a substance. This method may generally includeproviding a surface in contact with a droplet comprising a startingconcentration of the substance and having a starting volume; conductingone or more droplet operations to merge a wash droplet with the dropletto yield a combined droplet; and conducting one or more dropletoperations to divide the combined droplet to yield a set of dropletsincluding: (i) a droplet in contact with the surface having a decreasedconcentration and decreased quantity of the substance relative to thestarting concentration; and (ii) a droplet which is separated from thesurface.

The method of the invention may yield a droplet in contact with thesurface having a decreased quantity or substantially decreased quantityof the substance relative to the starting concentration. The resultingdroplet may in some embodiments have a volume which is approximately thesame as the starting volume. In some embodiments, the washing steps maybe repeated until a predetermined maximum quantity of the one or morecomponents is met or exceeded in the resulting droplet. Thepredetermined amount may represent a substantial reduction relative tothe starting concentration. In some cases, the resulting droplet may besubstantially free of the components. For example, in some embodiments,the reduction in amount exceeds 99, 99.9. 99.99, 99.999, 99.9999,99.99999, 99.999999 percent on a molar basis.

The method of the invention may yield a droplet in contact with thesurface having a decreased concentration or substantially decreasedconcentration of the substance relative to the starting concentration.The resulting droplet may in some embodiments have a volume which isapproximately the same as the starting volume. In some embodiments, thewashing steps may be repeated until a predetermined maximumconcentration of the one or more components is met or exceeded in theresulting droplet. The predetermined concentration limit may represent asubstantial reduction relative to the starting concentration. In somecases, the resulting droplet may be substantially free of thecomponents. For example, in some embodiments, the reduction inconcentration exceeds 99, 99.9. 99.99, 99.999, 99.9999, 99.99999,99.999999 percent.

8.6.1 Washing Beads

For protocols making use of beads, droplet with beads can be combinedusing droplet operations with one or more wash droplets. Then, whileretaining the beads (e.g., physically or magnetically), the mergeddroplet may be divided using droplet operations it into two or moredroplets: one or more droplets with beads and one or more dropletswithout a substantial amount of beads. In one embodiment, the mergeddroplet is divided using droplet operations into one droplet with beadsand one droplet without a substantial amount of beads.

Generally, each execution of a washing protocol results in retention ofsufficient beads for conducting the intended assay without undulydetrimental effects on the results of the assay. In certain embodiments,each division of the merged droplet results in retention of more than90, 95, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99,99.9. 99.99, 99.999, 99.9999, 99.99999, or 99.999999 percent of beads.In other embodiments, each execution of a washing protocol to achieve apredetermined reduction in the concentration and/or amount of removedsubstance results in retention of more than 99, 99.1, 99.2, 99.3, 99.4,99.5, 99.6, 99.7, 99.8, 99, 99.9. 99.99, 99.999, 99.9999, 99.99999, or99.999999 percent of beads. In still other embodiments, the amount ofretained beads is calculated and the results are adjusted accordingly.

In some embodiments, beads can be washed in reservoirs in which thebead-containing droplet and wash droplets are combined, beads areretained (for example by a magnet, by physical structures, electrostaticforces), and droplets lacking beads are dispensed from the reservoirusing droplet operations. For example, beads can be washed bydilute-and-dispense strategy whereby a wash buffer is added to thereservoir to dilute the contents, magnetically responsive beads arelocalized within the reservoir with a magnet and most of the solution isdispensed from the reservoir, and this cycle is repeated till acceptablelevels of washing are achieved.

8.6.1.1 Washing Magnetically Responsive Beads

A non-limiting example, illustrated in FIG. 11, involves immobilizingmagnetically responsive beads using a magnetic field. Immobilizedmagnetically responsive beads may be freed by reduction or eliminationof the magnetic field. Washing magnetically responsive beads maygenerally include the following steps:

-   (1) using droplet operations to position a droplet 1101 comprising    magnetically responsive beads 1102 and unbound substances 1103 in    proximity with a magnet 1104;-   (2) using droplet operations to combine a wash droplet 1106 with the    droplet 1101 comprising the magnetically responsive beads 1102;-   (3) immobilizing the beads 1102 by application of a magnetic field;-   (4) using droplet operations to remove some or all of the droplet    surrounding the beads to yield a droplet 1108 comprising the beads    with a reduced concentration of unbound target substance and a    droplet 1110 comprising unbound target substance;-   (5) releasing the beads 1102 by removing the magnetic field; (6)    repeating steps (2) to (3) or (2) to (4) until a predetermined    degree of purification is achieved.

In this manner, unbound substances, such as contaminants, byproducts orexcess reagents, can be separated from the beads. Each cycle produces adroplet including the beads but with a decreased level of the unwantedsubstances. Step (5) is not required in each washing cycle; however, itmay be useful to enhance washing by freeing contaminants which may betrapped in the immobilized beads. Steps may be performed in a differentorder, e.g., steps (2) and (3) may be reversed. Steps in the washingprotocol may be accomplished on a droplet microactuator using dropletoperations as described herein.

Another embodiment is illustrated in FIG. 12 and may comprise a topplate 1201, bottom plate 1202, electrodes 1203, and a magnet 1204. Theembodiment steps generally may include:

-   (1) using droplet operations to combine a slug 1205 with a droplet    1206 comprising magnetically responsive beads 1207 and unbound    material 1208 in proximity with magnet 1204;-   (2,3) with the beads 1207 immobilized, using droplet operations to    transport the resulting combined slug 1210 across the beads 1207 to    separate unbound material 1208 from the beads 1207;-   (4) using droplet operations to separate off a portion of the    combined slug 1210 to yield a portion 1212 comprising the beads with    a reduced concentration of unbound target substance and a portion    1214 comprising unbound target substance;

(5) repeating steps (1)-(4) as needed to achieve the desired reductionin unbound material.

In a related approach, the slug may be continuously supplemented byadding additional wash droplets and/or slugs as the slug is beingtransported across the immobilized beads. The process may continue untilthe desired reduction in unbound material is achieved.

FIG. 13 illustrates an alternative embodiment which may also comprise atop plate 1301, bottom plate 1302, electrodes 1303, and a magnet 1304.In this embodiment, the magnet 1304 is moved, such as in the directionof A1, to separate beads 1305 from unbound material 1306 in a combinedslug 1307 rather than moving the slug 1307. A similar approach involvesmovement of both the magnet and the slug to achieve separation (notshown). Yet another approach involves using multiple magnets to move thebeads (not shown).

In embodiments in which magnetically responsive beads are used, theinventors have found that application of a magnetic field though usefulfor temporarily immobilizing beads, moving beads and/or positioningbeads, sometimes results in unwanted aggregation of the beads. In oneembodiment, a surfactant is included to prevent or reduce beadaggregation. Examples of surfactants suitable for this purpose include:Tween® 20, Tween® 80, Triton X-100. Surfactants should be selected andused in amounts which reduce or eliminate bead aggregation and minimizenon-specific adsorption while at the same time not resulting insignificant loss of target analytes or reagents from the droplet.

Another approach to eliminating or reducing clumping aggregation ofbeads involves the use of smaller numbers of larger beads. Any number ofbeads which can be contained in a droplet during one or more dropletoperations may be used. In some embodiments, the number of magneticallyresponsive beads can range from 1 to several 100,000's. For example, inone embodiment, the invention makes use of one to 100 magneticallyresponsive beads per droplet. For example, the invention may make use of1, 2, 3, 4, 5, 6, 7, 8, 9, 10 . . . 100 magnetically responsive beadsper droplet. In one embodiment, the number of magnetically responsivebeads is from one to 10. Use of smaller numbers of magneticallyresponsive beads permits larger beads to be used. For example, in oneembodiment, the invention makes use of one to 100 magneticallyresponsive beads per droplet, where the beads have an average diameterof about 25 to about 100 microns. In another embodiment the inventionmakes use of one to 10 magnetically responsive beads per droplet, wherethe beads have an average diameter of about 50 to about 100 microns.

8.6.1.2 Washing Non-Magnetically Responsive Beads

A similar approach may be used with beads that are not magneticallyresponsive or not significantly magnetically responsive. As illustratedin FIG. 14, instead of using a magnetic field to immobilize beads 1401,a physical obstacle 1402 may be used to permit removal of some or all ofdroplet 1403 surrounding the beads 1401. The physical obstacle 1402 may,for example, include a membrane, sieve, and/or projection from thedroplet microactuator (e.g., from the top plate 1404 and/or bottom plate1405). Where a physical obstacle 1402 (projection or object) attached tothe top plate 1404 and/or bottom plate 1405 is employed, it should bearranged so as to permit transport using one or more adjacent electrodes1406 while preventing the beads 1401 from following, e.g., using aprojection from the top plate that leaves sufficient space for droplettransport and/or a projection with one or more openings that permits thedroplet to be transported through the opening while preventing the beadsfrom following.

8.6.2 Washing Droplet Microactuator Surfaces

FIG. 15 illustrates an example of an approach for washing a dropletmicroactuator surface. In this non-limiting example, a surface 1501 islocated on the interior of the top plate 1502. In this approach, (1) asample droplet 1503 including a target substance 1504 having affinityfor a surface component 1505 is (using droplet operations) brought intocontact with the surface 1501, causing (2) some portion or all of thetarget substance to be immobilized. (3) A wash droplet 1506 and thesample droplet 1503 are combined using droplet operations to yield acombined wash-sample droplet 1507. (4) The combined wash-sample dropletis then divided using droplet operations to yield a portion 1508 incontact with the surface and comprising a reduced concentration ofunbound target substance and a portion 1509 separated from the surfacecomprising unbound target substance. Steps (3) and (4) may be repeatedas needed to achieve the desired reduction in unbound material.

8.7 Cell Handling

Various protocols of the invention may make use of droplets includingcells. The droplets may include culture media for maintaining cellviability and/or growing cell cultures.

In some cases, the invention makes use of droplets having predeterminednumbers of cells. For example, in some embodiments, the invention maymake use of droplets including single cells. For example, droplets withsingle cells may be useful to product clonally pure cell populationsand/or to conduct experiments studying the reaction of single cells tospecific stimuli. Droplets with predetermined numbers of cells may beprovided by dispensing droplets from a cell suspension onto a droplettransport pathway or network from a suspension of cells and/or bydividing droplets with multiple cells into one or more subdroplets. Thesuspension may be supplied from an external source or may be stored in adroplet microactuator reservoir. Droplets can be analyzed to determinethe number of cells in each droplet, and droplets with a preselectednumber of cells can be routed downstream for further processing.Dispensed droplets with multiple cells may themselves be combined withone or more buffer droplets and divided into two or more sub-dropletsand analyzed for the presence of single cells.

Sort decisions can be based on droplet analysis. For example, lighttransmission may be used to identify droplets with a predeterminednumber of cells. Sort decisions may be made based on the measurement oftransmitted light. Other embodiments may employ automated image analysisand/or or multi-color fluorescence and/or scattering analysis. Dropletsnot meeting specifications can be reintroduced into the sample reservoirfor another attempt or transported to a waste reservoir.

Droplets meeting cell count specifications may be transported to dropletmicroactuator reservoirs and/or transported for sorting and/orenrichment. One approach to providing reservoirs with enriched cellcontent is illustrated in FIGS. 16A and 16B. In this embodiment,droplets 1602 are dispensed from a cell suspension 1604 and transportedbased on their characteristics to reservoirs 1606 on dropletmicroactuator 1600.

In other embodiments, droplets may be further manipulated, e.g., asdiscrete droplets for analysis of the cells contained within. Dropletsincluding predetermined numbers of cells may be used as inputs forvarious assay protocols described herein. In some embodiments, gravityis not used as the motive force for transporting droplets.

In one specific embodiment, tumor cells may be isolated on the dropletmicroactuator. Cells may, for example, be isolated from microliters offine-needle aspirates (FNA). In another embodiment, samples such asblood stem cells, bone marrow, GI washes, and cryopreserved-thawedsamples can be analyzed for cancer cells.

Immunogenic capture of relevant cells can be accomplished using antibodybeads, such as anti-cytokeratin beads, may be used to capture relevantcells from a sample prior to introduction into the droplet microactuatorand/or from a droplet on a droplet microactuator. Binding may beenhanced or incubation times reduced on the droplet microactuator byactively shuttling the droplet or vortexing the droplet within areservoir. Beads can be isolated and washed as described elsewhereherein. Target cells can be released into suspension in a droplet on thedroplet microactuator.

Uniform numbers of cells per droplet from can be dispensed from anon-chip reservoir using cell dispensing approaches described herein.Droplets with cells can be aliquoted into multiple on-chip reservoirs.Cells can be incubated in on-chip reservoirs. Cell viability can beassessed, e.g., using resazurin as a fluorescent redox indicator. Livingcells convert the non-fluorescent resazurin dye into resorufin whichfluoresces red. Non viable cells do not fluoresce. Cells can bedistributed to on-chip reservoirs and nucleic acid from the cells can beamplified using approaches as described herein.

8.8 Droplet Microactuator Architecture and Operation

The system of the invention generally includes a droplet microactuatorcontrolled by a processor. For example, the processor may, among otherthings, be programmed to control droplet manipulations on a dropletmicroactuator. A wide variety of droplet microactuator configurations ispossible. Various illustrations are provided in FIGS. 1, 2, 6, 9, and17. Examples of components which may be configured into a dropletmicroactuator of the invention include various filler fluids which maybe loaded on the droplet microactuator; fluid loading mechanisms forintroducing filler fluid, sample and/or reagents onto the dropletmicroactuator; various reservoirs, such as input reservoirs and/orprocessing reservoirs; droplet dispensing mechanisms; means forcontrolling temperature of the droplet microactuator, filler fluid,and/or a droplet on a droplet microactuator; and magnetic fieldgenerating components for manipulating magnetically responsive beads ona droplet microactuator. This section discusses these and other aspectsof the droplet microactuator and their use in the systems of theinvention.

8.8.1 Droplet Microactuator

The systems make use of a droplet microactuator. The dropletmicroactuator will include a substrate with one or more electrodesarranged for conducting one or more droplet operations. In someembodiments, the droplet microactuator will include one or more arrays,paths or networks of such electrodes. A variety of electrical propertiesmay be employed to effect droplet operations. Examples includeelectrowetting and electrophoresis.

In one embodiment, the droplet microactuator includes two or moreelectrodes associated with a substrate, and includes a means forpermitting activation/deactivation of the electrodes. For example, theelectrodes may be electronically coupled to and controlled by a set ofmanual switches and/or a controller. The droplet microactuator is thuscapable of effecting droplet operations, such as dispensing, splitting,transporting, merging, mixing, agitating, and the like. Dropletmanipulation is, in one embodiment, accomplished using electric fieldmediated actuation. Electrodes will be electronically coupled to a meansfor controlling electrical connections to the droplet microactuator.

The basic droplet microactuator includes a substrate including a path orarray of electrodes. In some embodiments, the droplet microactuatorincludes two parallel substrates separated by a gap and an array ofelectrodes on one or both substrates. One or both of the substrates maybe a plate. One or both substrates may be fabricated using PCB, glass,and or semiconductor materials as the substrate. Where the substrate isPCB, the following materials are examples of suitable materials: MitsuiBN-300; Arlon 11N; Nelco N4000-6 and N5000-30/32; Isola FR406,especially IS620; fluoropolymer family (suitable for fluorescencedetection since it has low background fluorescence); polyimide family.Various materials are also suitable for use as the dielectric componentof the substrate. Examples include: vapor deposited dielectric, such asparylene C (especially on Glass), and parylene N; Teflon AF; Cytop; andsoldermasks, such as liquid photoimageable soldermasks (e.g., on PCB)like Taiyo PSR4000 series, Taiyo PSR AUS series (good thermalcharacteristics for applications involving thermal control), andProbimer 8165 (good thermal characteristics for applications involvingthermal control); dry film soldermask, such as those in the DupontVacrel family; and film dielectrics, such as polyimide film (Kapton),polyethylene, and fluoropolymers like FEP, PTFE. Some or all of thesubstrate may also include a hydrophobic coating. Suitable examplesinclude Teflon AF; Cytop; coatings in the Fluoropel family; silanecoatings; fluorosilane coatings; and 3M Novec electronic coatings.

Were the droplet microactuator includes two plates, droplets may beinterposed in the space between the plates. Space surrounding thedroplets typically includes a filler fluid. The droplet microactuatorcan conduct droplet operations using a wide variety of fluid droplets,though conductive fluids are preferred.

Surfaces of the droplet microactuator are typically coated with ahydrophobic coating. For applications involving thermal cycling, ahydrophobic coating should be selected that is resistant to thermalstress during prolonged thermocycling operation. Examples of suitablethermal resistant materials include soldermasks such as Probimer® 8165which has been developed for use in the automotive industry and hasexcellent thermal shock resistance, and PCB board materials such asMitsui BN-300 which is resistant to high temperature and warpage.

Droplet transport occurs along a path or network of control electrodes.The array or path includes electrical connections for electricallycoupling electrodes to external circuitry. The array or path may alsoinclude electrical connections for electrically coupling certainelectrodes together. The electrodes are controlled via the externalcircuitry by a processor. Droplet operations may be effected bysupplying voltage to the electrodes. While the preferred voltage variesdepending on the thickness of the dielectric, for a dielectric constantin the range of 2-100 and thickness in the range of 1 nm to 10 mm, thepreferred energy per unit area limits are in the range of about 300microjoule/sq meter to about 300000 microjoule/sq meter. The preferredactivation voltage is in the range of about 1 mV to about 50 kV, orabout 1V to about 10 kV, or about 5V to about 1000V, or about 10V toabout 300V.

Typically, the electrodes are fired via a voltage relay. The dropletmicroactuator operates by direct manipulation of discrete droplets,e.g., using electrical fields. For example, a droplet adjacent to anenergized electrode with surrounding electrodes grounded will transportto align itself with the energized electrode, i.e., the droplet will betransported to the position of that electrode. A series of successivetransfers will transport droplets along the path or network of controlelectrodes. In addition to transport, other operations includingmerging, splitting, mixing and dispensing of droplets can beaccomplished in the same manner by varying the patterns of voltageactivation.

It should be noted that electrodes can be activated in a variety ofways. For example, an electrode can be activated by applying a DCpotential. Similarly, an electrode can be activated by applying an ACpotential, so that the activated electrode has an AC potential anunactivated electrode has a ground or other reference potential. Inanother aspect, the potential may be applied by repeatedly activating anelectrode and then inverting it. An AC mode can be effected by usingsoftware to rapidly switch between polarities of the outputs.

In some embodiments the invention employs droplet operation structuresand techniques described in U.S. Pat. No. 6,911,132, entitled “Apparatusfor Manipulating Droplets by Electrowetting-Based Techniques,” issued onJun. 28, 2005 to Pamula et al.; U.S. patent application Ser. No.11/343,284, entitled “Apparatuses and Methods for Manipulating Dropletson a Printed Circuit Board,” filed on Jan. 30, 2006; U.S. Pat. Nos.6,773,566, entitled “Electrostatic Actuators for Microfluidics andMethods for Using Same,” issued on Aug. 10, 2004 and 6,565,727, entitled“Actuators for Microfluidics Without Moving Parts,” issued on Jan. 24,2000, both to Shenderov et al.; U.S. Patent Publication No. 20060254933,entitled “Device for transporting liquid and system for analyzing”published on Nov. 16, 2006 by Adachi et al., the disclosures of whichare incorporated herein by reference for their teachings concerningstructures and techniques for conducting droplet operations.

Droplet operations can be rapid, typically involving average linearvelocities ranging from about 0.01 cm/s to about 100 cm/s, or from about0.1 cm/s to about 10 cm/s, more preferably from about 0.5 cm/s to about1.5 cm/s. Moreover, droplets may typically be manipulated at a frequencyof manipulation ranging from about 1 Hz to about 100 KHz, preferablyfrom about 10 Hz to about 10 KHz, more preferably from about 25 Hz toabout 100 Hz. In addition to being rapid, droplet manipulations usingthe droplet microactuator are also highly precise, and multiple dropletscan be independently and simultaneously manipulated on a single dropletmicroactuator.

Discrete droplet operations obviate the necessity for continuous-flowarchitecture and all the various disadvantages that accompany such anarchitecture. For example, near 100% utilization of sample and reagentis possible, since no fluid is wasted in priming channels or fillingreservoirs. Further, as noted above, droplet movement can be extremelyrapid. The droplet microactuator may in some cases be supplemented bycontinuous flow components and such combination approaches involvingdiscrete droplet operations and continuous flow elements are within thescope of the invention. Continuous flow components may be controlled bythe controller. Nevertheless, in certain other embodiments, variouscontinuous flow elements are specifically avoided in the dropletmicroactuator of the invention and/or methods of the invention. Forexample, in certain embodiments, one or more of the following componentsis excluded from a droplet microactuator and/or methods of theinvention: microchannels; fixed microchannels; networks ofmicrochannels; pumps; external pumps; valves; high-voltage supplies;centrifugal force elements; moving parts.

Electric field mediated actuation also obviates the need for otherdroplet operations and all the various disadvantages that accompany suchtechniques. It will be appreciated that the droplet microactuator maynevertheless be complemented or supplemented with other dropletmanipulation techniques, such as electrical (e.g., electrostaticactuation, dielectrophoresis), magnetic, thermal (e.g., thermalMarangoni effects, thermocapillary), mechanical (e.g., surface acousticwaves, micropumping, peristaltic), optical (e.g., opto-electrowetting,optical tweezers), and chemical means (e.g., chemical gradients). Whenthese techniques are employed, associated hardware may alsoelectronically coupled to and controlled by the controller. However, inother embodiments, one or more of these droplet operation techniques isspecifically excluded from a droplet microactuator of the invention.

The droplet microactuator can be manufactured in a highly compact formand can be driven using a very small apparatus. For example, dropletmicroactuator and apparatus may together be as small as several cubicinches in size. The droplet microactuator requires only small amounts ofelectrical power and can, for example, readily be operated usingbatteries. The droplet microactuator can perform droplet operationsusing extremely small droplets. Droplets are typically in the range offrom about 1 fL to about 1 mL, more preferably from about 100 pL toabout 1 μL, still more preferably from about 10 mL to about 1 μL.

The use of discrete droplets for on-chip processing instead ofcontinuous flows provides several important advantages. Since samplefluid need not be expended for priming of channels or pumps virtuallyall of the sample fluid can be used for analysis and very small volumesof sample (e.g., less than about 100 μL or less than about 50 μL or lessthan about 25 μL) can be analyzed. The same advantages apply to the useof reagents where reducing the volume of reagents consumed has theadvantage of reducing the cost of the analysis. The use of discretesmall-volume droplets also permits a large number of reactions toperformed in a small footprint (e.g. greater than 10 per cm² or greaterthan 100 per cm² or greater 1,000 per cm² or greater than 10,000 percm²).

Various components of the invention may be included as components of thedroplet microactuator. In fact, an entire system of the invention may beprovided as an integrated droplet microactuator. In some embodiments,the droplet microactuator includes various sensors and means forelectronically coupling the sensors to external circuitry. In otherembodiments, the droplet microactuator includes heaters and/or magneticfield generating elements and means for coupling such elements toexternal circuitry. Further, a droplet microactuator including any oneor more of the reagents described herein in a reservoir or in dropletform is also an aspect of the invention.

Optical windows can be patterned in the electrodes to enhance thecapability of performing optical detection on the chip. Where theelectrode is formed in an opaque material on a transparent substrate, awindow in the electrode can be created permit light to pass through thesubstrate. Alternatively, when the electrode material is transparent, amask can be created to eliminate stray light. Additionally, the openingcan be patterned as a diffraction grating. Adaptive optical windows canbe created as well, using a second electrowetting layer. For example,opaque oil (e.g. oil dyed black) can be used with a transparent dropletto create an temporary and movable optical window.

8.8.2 Cartridge

In some embodiments, the invention includes a cartridge for coupling tothe droplet microactuator. It will be appreciated that a cartridge,while not necessary to the operation of the invention, may be convenientin some circumstances. When present, the cartridge may include a meansfor electrically coupling the path or network of the dropletmicroactuator to a processor, e.g., a processor of a dropletmicroactuator system of the invention. In this embodiment, theelectrical connection is: electrodes—cartridge—processor, where theremay be additional elements between the three. In another embodiment, thecartridge may include means for physically coupling to the dropletmicroactuator. In this embodiment, the electrical connection may be:electrodes—processor—cartridge. Alternatively, the cartridge may lackelectrical components altogether.

When present, the cartridge may include reservoirs for one or morereagents, e.g., pre-loaded reagents. The droplet microactuator may beconfigured so that a fluid path may be established between the cartridgereservoirs and the interior of the droplet microactuator for flowingreagents, sample and/or filler fluid from the cartridge onto the dropletmicroactuator. For example, preloaded cartridge reservoirs may bedispensed into the droplet microactuator prior to, during, or aftercoupling of the cartridge to the analyzer. The cartridge may be sealed,self-contained and/or disposable. It may be supplied with or without adroplet microactuator. Such cartridges can be used to ensure repeatableassay conditions, permit safe handling and disposal of infectious orhazardous material, and/or reduce cross-contamination between runs. Thecartridge may, for example, include a machined plastic part. It may beaffixed to and provided in combination with the droplet microactuator.

The cartridge materials are selected to provide storage of reagentswithout degradation or contamination of the reagents. Moreover, theyshould be selected to provide reliable operation at elevated temperatureand to ensure compatibility with the real-time chemistry. They may, forexample, include molded plastic components. In some embodiments, sealed,disposable test cartridges enhance operator safety and facilitate safedisposal.

Various components of the droplet microactuator system may be includedon the cartridge. For example, present, the top-plate, which enclosesthe interior space of the droplet microactuator, may be provided as acomponent of the cartridge. Various sensors may also be included ascomponents of the cartridge.

8.8.3 Filler Fluid

The droplet microactuator of the invention includes one or more free(i.e. fluid-fluid) interfaces. Examples include a liquid-liquid orliquid-gas interface. Typically chemistry is performed in the primary(droplet) phase, and the secondary phase serves as a filler fluidseparating the droplets from each other. The secondary phase can, forexample, be a liquid, gel and/or a gas. Where the secondary phaseincludes a liquid, the liquid is sufficiently immiscible with theprimary liquid phase to permit the droplet microactuator to conduct oneof more droplet operations.

It should also be noted that the droplet microactuator may include morethan two phases. For example, in one embodiment the dropletmicroactuator operates based on an aqueous-oil-air three-phase system.In a related environment, the droplet microactuator may operate based onan aqueous-first oil-second oil three-phase system, such as a systemincluding an aqueous droplet surrounded by silicon oil, which is in turnsurrounded by a fluorosilicon oil. Generally, three-phase systems willinclude three components which are mutually immiscible or substantiallyimmiscible.

In another embodiment, oil or another immiscible liquid may be used as adroplet encapsulant for electrowetting. For example, a droplet can beencapsulated in a shell of oil by moving the droplet through an air/oilinterface. Each droplet would then have its own local bath of oil withthe space between encapsulated droplets filled with either air or athird immiscible liquid. Among other advantages, this approach is usefulfor minimizing the transfer of material between droplets in the systemby partitioning into the oil phase while retaining the advantageousproperties of the oil with respect to evaporation and fouling of thesurface. This approach may also be used to facilitate electrowetting ofnon-electrowettable liquids which are immiscible with electrowettableliquids. In a specific embodiment of this concept the immiscible liquidcan be chosen to be crosslinkable (by UV, heat, moisture or chemically)to create capsules of liquids with solid shells, for drug deliverysynthesis applications.

Further, in some applications it may be desirable or necessary toperform certain operations in an immiscible liquid, such as oil, andothers in air. The invention includes hybrid systems in which dropletmanipulation is performed both in air and in an immiscible liquid fillerfluid such as oil. For example, samples may be processed under oil andthen transported into an air-medium portion for evaporation forsubsequent analysis by MS. Conversely, a sample could be collected inair and then processed with droplets under oil. Thus, the dropletmicroactuator may include a transport path for moving droplets from adroplet microactuator surface in a space filled with filler fluid to adroplet microactuator open to the atmosphere or including a gaseousfiller fluid.

The filler fluid may be any fluid in which the droplet microactuatorcan, under the right conditions, conduct one or more droplet operations.It should be noted that certain filler fluids may be solids or highlyviscous fluids under certain conditions, e.g., during transport, whilethey are transformed into fluids for operation, e.g., by heating. Thefiller fluid may be a liquid or gas during operation of the dropletmicroactuator. Examples of suitable liquid filler fluids include,without limitation, silicone oils; fluorosilicone oils; hydrocarbons,including for example, alkanes, such as decane, undecane, dodecane,tridecane, tetradecane, pentadecane, hexadecane; aliphatic and aromaticalkanes such as dodecane, hexadecane, and cyclohexane, hydrocarbon oils,mineral oils, paraffin oils; halogenated oils, such as fluorocarbons andperfluorocarbons (e.g. 3M Fluorinert liquids); mixtures of any of theforegoing oils in the same class; mixtures of any of the foregoing oilsin different classes. Examples of suitable gas filler fluids include,without limitation, air, argon, nitrogen, carbon dioxide, oxygen,humidified air, any inert gases. In one embodiment, the primary phase isan aqueous solution, and the secondary phase is air or an oil which isrelatively immiscible with water. In another embodiment, the fillerfluid includes a gas that fills the space between the plates surroundingthe droplets. A preferred filler fluid is low-viscosity oil, such assilicone oil. Other suitable fluids are described in U.S. PatentApplication No. 60/736,399, entitled “Filler Fluids for Droplet-BasedMicrofluidics” filed on Nov. 14, 2005, the entire disclosure of which isincorporated herein by reference. The fluid may be selected to preventany significant evaporation of the droplets.

The phases of the fluids used in the protocols of the invention may beselected to facilitate protocols of the invention without undueformation of bubbles, loss of reagent to the filler fluid, and/oradherence of reagent to the droplet microactuator surface.

In certain embodiments of the invention the filler fluid may be selectedto reduce or prevent evaporation of sample, reagent, or other dropletsutilized in the protocols of the invention. The filler fluid may beselected to prevent sample, reagent, or other droplets utilized in theprotocols of the invention from evaporating and becoming too small forfurther effective manipulation. Similarly, the filler fluid can beselected to prevent evaporation of sample, reagent, or other dropletsutilized in the protocols of the invention from detrimentallyconcentrating species within the droplets in a manner which results inan unduly adverse affect on the intended use of the droplet. Moreover,the filler fluid may be selected to reduce or prevent transport ofmaterial from sample, reagent, or other droplets utilized in theprotocols of the invention across the phase boundary to maintain dropletvolume and/or ensure reliable microfluidic operation and/or assayresults. Miscibility between phases can sometimes result in shrinking(or swelling) of the droplet phase. To prevent or reduce this problem,one or more phases of the system may be saturated with the equilibriumconcentration of another phase to reduce shrinking or swelling. Thus,for example, the filler fluid may be saturated with the equilibriumconcentration of the solvent for sample, reagent, or other dropletsutilized in the protocols of the invention, and/or one or more of thesample, reagent, and/or other droplets utilized in the protocols of theinvention may be saturated with the equilibrium concentration of thefiller fluid.

In some embodiments, a liquid filler fluid is selected to minimizecontact between the droplet and droplet microactuator surfaces. That is,a film of liquid may exist between the droplet and surface whichprevents material within the droplet from coming into contact with andadhering to the coated surface. This approach helps to prevent foulingof the surface and related interference with droplet transport. Forexample, it has been observed that high concentrations of certainproteins in water droplets readily stick to certain hydrophobic surfacesspoiling the hydrophobic nature of these surfaces; whereas, the samedroplets can be moved across the same surfaces without appreciableadhesion of proteins if bathed in an oil which minimizes contact betweenthe two surfaces. This approach may also help to avoidcross-contamination between droplets caused by deposition of materialfrom one droplet which is then picked up by a second droplet. In asimilar embodiment, a film between the droplet and droplet microactuatorsurface can be used to lubricate the droplet by preventing friction-likephysical interactions between the droplet and surface during dropletoperations.

In one embodiment, the invention provides a thin coating of a liquidfiller fluid layer in an otherwise gas filled system. For example, theinvention provides a microfluidic system including an open or enclosedsystem including a thin layer of filler fluid, such as oil, layered on adroplet microactuator surface, wherein the system is otherwise filledwith a gas. The oil is of sufficient thickness to provide lubricationand contamination of droplet microactuator surfaces and contamination ofdroplets via droplet microactuator surfaces. Preferably the oil isselected to minimize transport of material between the droplet and oilphases. One advantage of this approach is reduction of carry-over in thedroplet microactuator. The surface may in some embodiments be treated bycoating it with the filler fluid while operating in air. This approachis also useful for loading operations as a means to retain thelubricating effect of oil while avoiding trapping of oil bubbles in thebulk filler fluid.

Treatment of a Teflon AF surface with silicone oil can provide some ofthe lubrication benefit of silicone oil filler fluid even when operatingin air. This approach can be used to prime the droplet microactuatorwith a lubricating layer of oil, followed by replacement with air toallow samples to be loaded without introduction of bubbles, followed byre-introduction of oil to prevent evaporation of the samples. Thus thebenefits of each kind of system are available depending on the type ofmicrofluidic processing to be carried out.

In another embodiment, the filler fluid can be completely exchanged atdifferent steps within a protocol. For example, a gas filler fluid canbe introduced during sample loading to prevent trapping of air bubblesand then a liquid filler fluid can be pumped in to prevent evaporationof the liquid. Different types of filler fluid can be pumped into or outof the system depending on the particular assay steps to be performed.

In yet another embodiment, multiple filler fluids can be used within asingle system. For example, a droplet microactuator can be selected tohave separate gas filled and liquid filled regions. Operations orcertain types of droplets can be segregated between the different fillerfluid regions.

The filler fluid may be selected based on its refractive index to eithermatch the droplet to prevent refraction of light passing through or nearthe droplet. Alternatively the filler fluid may be selected with arefractive index that differs from the droplet to provide contrast forcertain types of optical measurements or optical manipulations. A fillerfluid may be chosen to have a lower index of refraction than the primaryliquid so that light can be transmitted though the primary liquid bytotal internal reflection. The primary phase can include highlyelongated droplets which can serve as “light pipes” to convey lightbetween two locations, e.g. to facilitate optical analyses.

The filler fluid may be selected based on its color to facilitatesdirect or indirect visualization of the droplet, e.g., by providingcontrast between the sample, reagent, and/or other droplets used in theprotocols of the invention and the filler fluid. This approach canenhance visualization of the different phases, for example todistinguish droplets from filler fluid or from air bubbles. In opticalapplications, the differential absorbance of the two phases can be usedto modulate the color of light passing through the system. As anotherexample, in applications where fluorescence measurements are made withindroplets it may desirable for the oil to include molecules, such asdyes, that absorb the emitted wavelength of light to minimize cross-talkbetween reactions occurring in adjacent droplets.

The filler fluid may be selected to have particular thermal propertiesthat can either thermally insulate the droplets or conduct heat awayfrom the droplets. For example, in the amplification protocols of theinvention, a thermally conductive or low heat capacity filler fluid maybe desirable to permit rapid changes in temperature. For applicationswhere a steady temperature is required a thermally insulating or highheat capacity filler fluid can be used to provide temperature stability.

The filler fluid may be selected to undergo a phase change uponpresentation of an appropriate stimulus. For example, a wax-like fillerfluid (e.g. paraffin wax or octadecane) can be used where the fillerfluid is changed from solid to liquid form by application of heat.Lowering the temperature would return the filler fluid to a solid sothat droplets would be contained within a solid matrix. Encapsulation ofthe liquid phase within a solid may facilitate storage and handling ofthe sample, reagent, and/or other droplets utilized in the protocols ofthe invention and/or allow for safe and convenient disposal of thematerials following use of the droplet microactuator. The filler fluidcan be stored as a solid on the droplet microactuator, in acartridge-based reservoir, or elsewhere, and heated to permit the fluidto flow into and fill the droplet microactuator. Or the immisciblefiller fluid can be selected to be crosslinkable (by UV, heat, moistureor chemically) to create capsules of liquids within a solid shell.

The filler fluid may be selected to have particular gas permeability orsaturation properties. In certain applications a reaction occurringinside the droplet may consume oxygen or other gas which may need to bereplenished by gas contained within or transported through the fillerfluid. For example, some fluorinated oils have useful gas permeabilityproperties for such applications. Alternatively, the filler fluid may beselected to exclude certain gases from the droplet, for example tomaintain anaerobic conditions within the droplet. The filler fluid maybe selected to have a certain degree of miscibility or partitioning intothe droplet phase. Usually, complete or substantially complete lack ofmiscibility between the droplet and filler fluid is desired, but someapplications may benefit from some limited degree of miscibility betweenthe phases or partitioning of particular molecules between the phases,e.g., liquid-liquid extraction applications. In certain applicationswhere dissolved gases in the filler fluid may be problematic, a meansfor degassing the filler fluid prior to or during use may need to beprovided. For example, filler fluid may be degassed by incubation undervacuum, heating, sparging or by centrifugation.

The filler fluid may be selected to have a particular surface orinterfacial tension with the droplet phase or with the dropletmicroactuator surfaces. Surfactants can be added to the filler fluid tostabilize liquid films that may be present between the droplet and solidphases. Examples of suitable surfactants include nonionic low HLB(hydrophile-lipophile balanced) surfactant. The HLB preferably less thanabout 10 or less than about 5. Suitable examples include: Triton X-15(HLB=4.9); Span 85 (HLB 1.8); Span 65 (2.1); Span 83 (3.7); Span 80(4.3); Span 60 (4.7); and fluorinated surfactants.

Surfactants are preferably selected and provided in an amount which (1)results in more droplet operations on the droplet microactuator ascompared to corresponding droplet microactuator without the surfactant;or (2) makes one or more droplet operations possible on the dropletmicroactuator as compared to corresponding droplet microactuator withoutthe surfactant; or (3) makes one or more droplet operations morereliable on the droplet microactuator as compared to correspondingdroplet microactuator without the surfactant. In a related example,surfactants are preferably selected and provided in an amount whichmakes one or more droplet operations possible or more reliable fordroplets including one or more specific reagents or mixtures on thedroplet microactuator as compared to droplet operations for the samedroplets including one or more specific reagents or mixtures on acorresponding droplet microactuator without the surfactant. In anotherrelated example, surfactants are preferably selected and provided in anamount which makes one or more droplet operations possible or morereliable for one or more droplets including amphiphilic molecules on thedroplet microactuator as compared to droplet operations for the samedroplets including amphiphilic molecules on a corresponding dropletmicroactuator without the surfactant.

In a preferred embodiment, the surfactant is added to the filler fluidin an amount which ranges from about 0.001 to about 10% w/w, or about0.001 to about 1% w/w, or about 0.001 to about 0.1% w/w. For example, inone embodiment the filler fluid is 2 cSt silicone oil and the surfactantis Triton X-15 in an amount which ranges from about 0.001 to about 10%w/w, or about 0.001 to about 1% w/w, or about 0.001 to about 0.1% w/w.The solid-liquid interfacial tension may be adjusted to control thewetting of the filler fluid on the droplet microactuator surfaces, forexample, to control the formation, thickness or behavior of thin filmsof the filler fluid between the droplet and droplet microactuatorsurfaces or to control the wetting behavior of the fluid when filling oremptying it from the droplet microactuator.

By doping filler fluid with surfactant, the inventors have discoveredthat it is possible to increase the concentrations of compatible proteinsolutions by more than 3 orders of magnitude, from mg/L to mg/mL. Theinventors were able to reliably dispense and transport 25 mL droplets of75 mg/mL lysozyme solution using the new filler fluid. For example, thefiller fluid may be a silicone oil doped with a surfactant, such asTriton X-15. Preferably the surfactant is a lipophilic surfactant. Inone embodiment, we added 0.1% (w/w) Triton X-15, a lipophilicsurfactant, to the oil so that high concentrations protein dropletscould be formed or dispensed from on-chip reservoirs. Droplet transportfor all compatible fluids is fast (typically about 3-10 cm/sec) andreliable (>25,000 operations). In one embodiment, the filler fluidincludes a surfactant dopant in an amount which results in an increasein the concentration of a protein that can be reliably dispensed on thedroplet microactuator.

The filler fluid may be selected to have a particular viscosity orvolatility. For example, a low viscosity liquid (e.g. 0.65 cSt. Siliconeoil) facilitates transport of droplets while a low volatility fillerfluid (e.g., 2, 5 or 10 cSt. Silicone oil) may be desirable to preventloss of filler fluid by evaporation, particularly in nucleic acidamplification applications performed at elevated temperature. In someapplications, evaporation of the filler fluid can be desired, so a lowvolatility filter fluid may be selected. The filler fluid may beselected to have a particular viscosity dependence on temperature, sincethe viscosity of the filler fluid affects the fluid dynamics and thetemperature on the droplet microactuator may vary. In nucleic acidamplification protocols of the invention, the filler fluid is selectedso that any viscosity changes resulting from thermal cycling are notunduly detrimental to conducting droplet operations required foreffecting the amplification protocols.

The filler fluid may be selected to have particular electricalproperties. For example, certain applications including electrowettingfavor the use of a filler fluid that is non-conductive (e.g., siliconeoil). Or the dielectric permittivity can be selected to control thecoupling of electrical energy into the system from external electrodes.In certain applications a non-conductive filler fluid can be employed asan electrical insulator or dielectric in which the droplet floats justabove the electrodes without physically contacting them. For example, inan electrowetting system a layer of filler fluid (e.g., silicone oil)between the droplet and electrode can be used to provide electrostaticcontrol of the droplet. Filler fluids may be deionized to reduceconductivity.

The filler fluid may be selected to have a particular density relativeto the droplet phase. A difference in density between the two phases canbe used to control or exploit buoyancy forces acting upon the droplets.Examples of two-phase systems useful in this aspect of the inventioninclude water/silicone oil, water/flourinert, and water/fluorosiliconeoil. When one phase is buoyant, then that effect can be exploited in avertical configuration as a means to transport one phase through theother. For example, a waste or collection well can exist at the top orbottom of the droplet microactuator where droplets are delivered to thatreservoir by simply releasing them at an appropriate point and allowingthem to float or sink to the target destination. Such an approach may besuitable for use in removing reactant from a droplet microactuator, e.g.removing fluid containing amplified nucleic acid for use in otherprocesses. Density differences can also be used as a means to control orengineer contact between the droplets and droplet microactuatorsurfaces. For example, a droplet not normally contacting a top-plate canbe released to sink or float to that surface to contact it. Densitydifferences and buoyancy effects can also be exploited for sensingapplications in which the movement of droplets is detected and relatedto a change in position, orientation or acceleration.

The filler fluid is selected for material compatibility with the dropletmicroactuator surfaces. For example, certain filler fluids can etch,dissolve, contaminate, absorb into or otherwise be incompatible withcertain droplet microactuator materials. For example, fluorinatedhydrocarbons, such as Fluorinert, may be incompatible with Teflon AF orCytop surfaces because of their tendency to dissolve these materials,while silicone oils may be incompatible with PDMS surfaces due to thetendency of these materials to dissolve each other.

The filler fluid is selected for biochemical compatibility with sampleand reagents used in the protocols of the invention.

The invention may include means for controlling the introduction orcirculation of the filler fluid within the droplet microactuator,cartridge and/or system. In one mode of operation the filler fluid isinjected once during the initialization of droplet microactuatoroperation. The filler fluid may be provided from an external sourceusing a syringe, dropper, pipettor, capillary, tube or other means.Alternatively, the filler fluid may be provided from a reservoirinternal to the droplet microactuator assembly or cartridge. As anexample, the fluid can be contained within a sealed pouch which ispunctured or compressed to transfer the liquid into the dropletmicroactuator.

In another mode of operation a means can be provided for multipleintroductions or recirculation of one or more filler fluids within thedroplet microactuator. A secondary fluid-handling system can be providedto inject and to remove fluid from within the droplet microactuator.Pressure, gravity or other means such as the use of thermal gradientscan be used to transport the filler fluid into or out of the dropletmicroactuator. Such a system can be used for the following purposes:

-   (1) To replenish filler fluid lost to evaporation or leakage over    time. A slow steady flow or periodic injection of filler fluid can    be employed to make up for any loss of filler fluid volume.-   (2) To provide “clean” filler fluid either continually or    periodically to reduce contamination between droplets. The filler    fluid can be cleaned either by completely replacing it or by    circulating it through a filter or bed of absorbent material    selected to remove contaminants.-   (3) To provide a means for transporting droplets to waste. For    example, at the end of an assay, droplets can be released and    allowed to flow with the filler fluid to the outlet providing a    means to “flush” the droplet microactuator. Flushing the droplet    microactuator can be performed to reset the status of the droplet    microactuator in preparation to perform additional assays.-   (4) To exchange the filler fluid when different fluids may be    desired for certain steps, for example to replace oil with air to    allow drying of droplets, or to replace one oil with a different    oil.-   (5) To provide a means of controlling the temperature of the    droplets by heating or cooling the fluid as it is circulated through    the droplet microactuator. For example, PCR can be performed in    droplets containing the appropriate PCR reagents (e.g., primers,    nucleotides, and polymerase) by circulating temperature controlled    filler fluid through the droplet microactuator to perform    thermocycling. The temperature of the filler fluid entering and    leaving the droplet microactuator can be directly measured and the    temperature and flow rate of the filler fluid can be adjusted to    provide optimal temperature control inside the droplet    microactuator.

Local regions of filler fluid or even individual units of filler fluidfor each droplet can be used. For example aqueous droplets can beencapsulated in an individual shell of fluid, such as oil, which movesalong with that droplet. Each such droplet would then have its own localfluid bath with the space between encapsulated droplets filled withthird immiscible liquid such as air or fluorosilicone oil. This approachcan be used to minimize the transfer of material between droplets in thesystem by partitioning into the oil phase while retaining theadvantageous properties of the oil with respect to evaporation andfouling of the surface. The shells of oil can be created by simplymoving the droplet through an oil interface, pinching off a unit of oilas the droplet creates a bulge along the interface.

Hybrid systems can be implemented in which different regions of thedroplet microactuator are filled with different fluids. For example,samples can be processed under oil and then transported into an airportion to be evaporated for subsequent analysis by MS. Conversely, asample can be collected in air and then processed under oil.

Magnetically responsive beads can be used to move material between oiland water phases on a droplet microactuator. Generally, water-solublecompounds or materials tend to remain within the droplets, unable tocross the oil-water meniscus in significant quantities, and oil-solublecompounds or materials remain in the lipophilic filler fluid. When thematerial is attached to magnetically responsive beads, a magnetic fieldmay be used to move the beads and attached material across the oil-waterboundary. The beads need to be selected such that they have sufficientaffinity for oil and water so that they can readily cross the meniscus.This operation is useful for drying or concentrating materials and canalso be used to facilitate washing and/or dilution. For example,material bound to a magnetically responsive bead can be removed from onedroplet and transferred by way of the filler fluid to another droplet.

Filler fluid can be circulated through the droplet microactuator toreduce contamination during and/or between runs. Filler fluid can becontinually or periodically flowed through the droplet microactuator, sothat fresh filler fluid is constantly supplied to the dropletmicroactuator. In addition to removing contaminates contaminated oil,this technique could be used at the end of a run to clear droplets fromthe array by removing the voltage so that droplets are released and flowwith the oil to an exterior of the droplet microactuator and/or into awaste reservoir.

8.8.4 Droplet Microactuator Loading

The droplet microactuator generally includes one or more input ports forthe introduction of one or more filler fluids, reagents and/or samples(e.g., reagents and/or samples for conducting protocols and/or assays asdescribed elsewhere herein, e.g., in Sections 8.1, 8.2, 8.3, 8.4 and/or8.5) into the droplet microactuator. In some embodiments, samples orreagents are loaded via the input ports using conventional robotics. Inone alternative embodiment, droplets of sample or reagent are separatedby plugs of oil in a long pre-loaded glass capillary which whenconnected to the droplet microactuator allows droplets of sample orreagent to be captured and routed on the droplet microactuator as theyare pumped out of the capillary into the input port. Another loadingtechnique involves pre-stamping reagents onto the droplet microactuatorand allowing them to dry, e.g., using a high-speed reagent stamping orprinting process. Yet another approach involves the use of a directplate-to-droplet microactuator interface in which the contents ofplates, e.g., 1536 or 384 or 96 well plates, are transported onto thedroplet microactuator in parallel by using pressure to force thecontents through input ports aligned with wells. Loading hardware may insome embodiments be electronically coupled to and controlled by thecontroller.

8.8.5 Reservoirs

The droplet microactuator includes various reservoirs, such as inputreservoirs and/or processing reservoirs.

8.8.5.1 Input Reservoirs

In some embodiments, the droplet microactuator includes one or moreinput reservoirs in fluid communication with one or more input ports,typically in direct fluid communication with the input ports. The inputreservoir(s) serve as reservoirs for storage of bulk source material(e.g. reagents or samples) for dispensing droplets (e.g. reagentdroplets or sample droplets). Thus, the input reservoir(s) may, forexample, serve as sample wells or reagent wells.

The input reservoirs generally include one or more well walls definingan interior space and an opening. The interior space defined by the wellwalls is at least partially isolated by the well walls from theremainder of the interior of the droplet microactuator. The well may beadjacent (in any direction, e.g., vertically or laterally) to a portsuitable for introduction of fluid from an exterior of the dropletmicroactuator into the input reservoir. One or more openings in the wellwalls may be provided to enable fluid communication with the interiorvolume of the droplet microactuator for dispensing of droplets into thisinterior volume. The opening(s) may permit fluid to flow or betransported into the interior volume of the droplet microactuator ontothe path or network of electrodes. Input reservoirs may also include oneor more vents for permitting displacement of filler fluid from the inputreservoir as fluid is introduced into or removed from the well via theport or the opening.

The input reservoirs may further include one or more planar controlelectrodes in a top or bottom plate adjacent to or within the spacedefined by the well walls. The planar electrodes are electronicallycoupled to and controlled by the controller. In a preferred embodiment,the planar electrode has two or more branches or rays, such thatactivation of the control electrode during droplet dispensing in thepresence of a fluid exerts a “pull” on the fluid in a direction which isgenerally opposite to the direction of droplet dispensing. In somecases, the shape of the electrode results in a multi-vector pull havinga mean vector which has a direction generally opposite to the directionof the droplet being dispensed.

Well walls may, for example, be formed by protrusions from the top orbottom plates, and/or may be formed by deposition of a wall-formingmaterial on a surface of the top or bottom plate. For example, wellwalls may be formed from a soldermask material or polymeric gasketmaterial deposited and patterned on the surface. In some embodiments asource of continuous or semi-continuous sample or reagent flow iscoupled in fluid communication with one or more of the input ports.

It should be noted that while droplet dispensing may be conducted fromdefined reservoirs, in some embodiments, droplet dispensing is conductedwithout the use of physically defined reservoirs. Dispensing may proceedfrom source droplet which is confined during droplet dispensing, e.g.,by electrowetting forces or by hydrophilic surfaces.

8.8.5.2 Processing Reservoirs

The droplet microactuator may also include one or more processing areasor reservoirs. These areas or reservoirs serve as a location forexecuting various droplet processing steps, such as mixing, heating,incubating, cooling, diluting, titrating, and the like. The dropletmicroactuator includes one or more paths or networks of controlelectrodes sufficient to transport droplets from the one or more inputports to the one or more processing areas or reservoirs. In some casesthe processing areas are simply components or sections of these paths ornetworks. In other embodiments, the processing areas are definedprocessing reservoirs. Such reservoirs may, for example, be structuredgenerally in the same manner as the input reservoirs described above.However, the processing reservoirs are typically not in direct fluidcommunication with the input ports, i.e., droplet transport along theone or more paths or networks of control electrodes is required addreagent or sample to the processing reservoir(s). In some cases, theprocessing reservoirs include a path or network of reservoirs therein topermit droplet operations within the processing reservoirs.

8.8.5.3 Droplet Operations

The droplet microactuator may conduct various droplet operations withrespect to a droplet. Examples include: loading a droplet into thedroplet microactuator; dispensing one or more droplets from a sourcedroplet; splitting, separating or dividing a droplet into two or moredroplets; transporting a droplet from one location to another in anydirection; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; cooling a droplet;disposing of a droplet; transporting a droplet out of a dropletmicroactuator; other droplet operations described herein; and/or anycombination of the foregoing.

Droplet dispensing refers to the process of aliquoting a larger volumeof fluid into smaller droplets. Dispensing is usefully employed at thefluidic interface, the input reservoirs, and at processing reservoirs.Droplets may be formed by energizing electrodes adjacent to the fluidreservoir causing a “finger” of fluid to be extended from the reservoir.When the fluid front reaches the terminal electrode, the intermediateelectrodes are de-energized causing the fluid to retract into thereservoir while leaving a newly-formed droplet on the terminalelectrode. As previously noted, one or more electrodes in the reservoirmay also be energized to assist in separating the droplet beingdispensed from the bulk fluid. Because the droplet conforms to the shapeof the electrode, which is fixed, excellent accuracy and precision areobtained. Droplet dispensing is controlled by the controller. In someembodiments the invention employs droplet dispensing structures and/ortechniques described in U.S. Pat. No. 6,911,132, entitled “Apparatus forManipulating Droplets by Electrowetting-Based Techniques,” issued onJun. 28, 2005 to Pamula et al.; U.S. patent application Ser. No.11/343,284, entitled “Apparatuses and Methods for Manipulating Dropletson a Printed Circuit Board,” filed on filed on Jan. 30, 2006; U.S. Pat.Nos. 6,773,566, entitled “Electrostatic Actuators for Microfluidics andMethods for Using Same,” issued on Aug. 10, 2004 and 6,565,727, entitled“Actuators for Microfluidics Without Moving Parts,” issued on Jan. 24,2000, both to Shenderov et al., the disclosures of which areincorporated herein by reference.

In some embodiments, droplet operations are mediated by electrowettingtechniques. In other embodiments, droplet operations are mediated byelectrophoresis techniques. In still other embodiments, dropletoperations are mediated by electrowetting techniques and byelectrophoresis techniques.

In one embodiment, separations may be performed using a combination ofelectrowetting and electrophoresis. Electrowetting microactuation can beused to create a channel to perform electrophoresis; to deliver a sampleto the channel or capture a sample fraction from channel following anelectrophoretic separation. For example, for forming a channel,electrowetting can be used to deform (stretch) a droplet of separationmedium in a long thin shape followed. In some cases, the channel may bepolymerized, e.g., using UV polymerization. In other cases, the channelmay be formed by using droplet operations to add droplets into aphysically confined microchannel. In a related embodiment, the effectivelength of an electrophoresis channel can be increased by capturing thefraction of interest in a droplet at the output and then returning it tothe input in a cyclical fashion. Using the same principle, a series ofprogressively finer separation can be performed. Separations may also beaccomplished using multiple different separation mediums at the sametime.

Droplet splitting or dividing of droplets generally involves separatinga droplet into two or more sub-droplets. In some cases, the resultingdroplets are relatively equal in size.

Transporting involves moving a droplet from one location to another inany direction. Droplets may be transported on a plane or in threedimensions. It will be appreciated that a variety of droplet operations,such as dispensing and/or splitting may include a transporting element,in which on droplet is transported away from another droplet.

Merging involves combining two or more droplets into a single droplet.In some cases, droplets of relatively equal size are merged into eachother. In other cases, a droplet may be merged into a larger droplet,e.g., combining droplet with a larger volume present in a reservoir.

Mixing a droplet involves various droplet manipulations, such astransporting or agitating, that result in a more homogenous distributionof components within the droplet. In one mixing embodiment, a dropletpositioned over an electrowetting electrode is rapidly and cyclicallydeformed in place by activating and deactivating the electrode, inducingfluid currents within the droplet which facilitate mixing.Frequency-dependent effects such as mechanical resonances may be used totune the quality and speed of mixing. Compared to techniques whichrequire transport of droplets on a surface for mixing this approachminimizes the area required for mixing. This mixing scheme can beemployed without the presence of a top plate. Due to space-savingadvantage, this scheme could provide for simplified mixing in reactionwells since only one electrode is needed.

Reagents or samples from reservoirs may be dispensed as discretedroplets for transport to other locations on the droplet microactuator.

The invention includes droplet operations using droplets comprisingbeads. A variety of such operations are described elsewhere herein. Inone embodiment, beads are used to conduct droplet operations on reagentsthat are prone to interfere with droplet operations. For example,certain proteins may be prone to bind to surfaces of a dropletmicroactuator and/or to partition into the filler fluid. Immobilizingsuch compounds on hydrophilic beads can be used to facilitate dropletoperations using the compounds. The compounds can be bound to the beads,and the beads can contained with a droplet which is subjected to dropletoperations.

In one particular dispensing operation, coagulation is used to separateserum from whole blood. Whole blood is loaded onto the chip and combinedwith a droplet comprising a coagulating agent. Following coagulation,droplets are dispensed from the sample. Because cells and platelets aretrapped in place, the liquid dispensed from the sample will contain onlyserum.

8.8.6 Thermal Control

The droplet microactuator of the invention may include a means forcontrolling the temperature of the droplet microactuator or a region ofthe droplet microactuator. Among other things, thermal control is usefulfor various protocols requiring heating or cooling steps. Examplesinclude amplification protocols requiring thermal cycling and variousassays that require incubation steps.

8.8.6.1 Thermal Control Designs

In general, thermal control may be provided in three ways: (1) thermalcontrol of the entire droplet microactuator; (2) thermal control of aregion of a droplet microactuator using a heater that is in contact withor in proximity to the controlled region; and (3) thermal control of aregion of the droplet microactuator using a heater that is integratedinto the droplet microactuator (e.g., in the substrate comprising thepath or array of electrodes and/or in a top plate of the dropletmicroactuator, when present). Combinations of the foregoing approachesare also possible. Two approaches previously discussed are illustratedin FIG. 2.

In an integrated heater approach, temperature zones can be created andcontrolled using thermal control systems directly integrated into thedroplet microactuator. Integration of thermal control through thin-filmheating elements fabricated directly on the droplet microactuator isalso useful to maximize the speed, throughput and quality ofamplification reactions on the droplet microactuator. Due to their smallthermal mass, droplets can be thermally cycled extremely rapidly.Thermal control is enhanced by locating the heating elements proximateto the droplets and reducing the parasitic thermal losses between theheater and the droplet. Heating elements can be integrated into the topplate and/or bottom plate of the droplet microactuator.

Integrating heating elements onto the droplet microactuator also enablesthe use of multiple distinct thermal zones within the dropletmicroactuator. This permits multiple steps in an analysis, such assample preparation and thermal cycling, requiring different temperaturesto be performed simultaneously on different portions of the dropletmicroactuator. Droplets can be physically transported or “shuttled”between zones of different fixed temperatures to perform the thermalcycling aspects of the amplification reaction. This approach can produceeven faster reactions, since heating and cooling of the entire thermalzones is no longer rate-limiting. Instead, heating and cooling rates aredetermined by the time required to transport the droplets between thezones and the time required for the droplet temperature to equilibrateto the temperature of the zone once it arrives within the zone, both ofwhich are expected to be very fast. A further advantage is that reactionsteps can be “queued” rather than “batched” to permit greateroperational flexibility. For example, discrete samples can becontinuously fed into the droplet microactuator rather being deliveredat a single point in time.

Droplets may be thermally cycled in batch mode using a single heater orin flow-through mode by circulating the droplets through distincttemperatures zones created by the heating elements. The essentialdifference between batch and flow-through modes is that in batch modethermal control is effected by varying the temperature of the heaterwhile in flow-through mode, thermal cycling is effected by transportingthe droplets among distinct constant temperature zones. In the “batch”method a single integrated thin-film heater on the droplet microactuatorwas used to thermally cycle static droplets located within the heaterzone. In the “flow-through” method, two distinct fixed temperature zoneswere created on the droplet microactuator and thermal cycling wasperformed by shuttling the droplets between the two zones.

In the “batch” case, the thermal mass of the heater itself as well asthermal losses may be minimized through the use of thin-film heatersplaced directly adjacent to the droplets. Because the thermal masses,including the droplet itself, are so small, rapid temperature changescan be effected. Passive cooling (in filler fluid) is also rapid becausethe total energy input into the system is extremely small compared tothe total thermal mass.

For “flow-through” heating, a larger thermal mass is desirable becauseit helps to stabilize the temperature while a slower ramp rate istolerable because the heater temperature is not varied once it reachesits set point. A flow-through system can, for example, be implementedusing block heaters external to the droplet microactuator which weremore accurate and easier to control than thin-film heaters although, inprinciple either type of heater could be used to implement eithermethod.

In another embodiment, temperature is controlled by flowing orrecirculating heated filler fluid through the chip and around thedroplets.

The droplet microactuator layout is scalable, such that a dropletmicroactuator may include a few as one heating zone up to tens, hundredsor more heating zones.

8.8.6.2 Heater Types

Heaters may be formed using thin conductive films. Examples of suitablethin films include Pt heater wires and transparent indium-tin-oxide(ITO). ITO provides better visualization of the droplets for real-timeobservation. A remotely placed conventional thermocouple (TC) fortemperature regulation can also be used. In one embodiment, tiny metal(e.g., copper) vias in the PCB substrate are used to create tightthermal junctions between the liquid and the remote TC. Further, sampletemperature can be determined by monitoring the copper via using asurface mount thermistor or an infrared sensor. One advantage of using athermistor is that they are small enough (2×2 mm) to be soldereddirectly on the droplet microactuator, while an advantage of using IR isthat it is non-contact method which would simplify the interfacing.Because the thermal conductivity of copper is at least 700 times greaterthan the FR-4 substrate (350-390 W/m·K versus 0.3-0.5 W/m·K) thetemperature of a Cu via will accurately represent the temperature insidethe liquid. Heaters may be integrated on the bottom and/or top (whenpresent) plate of the droplet microactuator and on the bottom and/or topsurface of either plate, or integrated within the structure of eitherplate.

In one flow-through embodiment, reduced thermal gradients can beprovided by using heaters to create a continuous temperature gradientacross the droplet microactuator (e.g., from 100 to 50° C.). The use ofa continuous gradient will eliminate the need to overcome the steeptemperature gradients found along the edge of the heater blocks. Acontrolled temperature gradient would also significantly enhance thefunctionality of the device by allowing protocols with arbitrary numbersof temperature points to be implemented. Furthermore, each reaction canbe performed with a custom thermal protocol while only the temperaturesof the two or more blocks would need to be thermally regulated. Thedroplets will be transported to and held at the appropriate locationbetween the heaters to achieve a target temperature. The fluorescence ofthe droplets can be imaged using a fluorescence sensor as they aretransported over a detection spot. The temperature of the upper andlower target temperatures can be varied by changing the location of thedroplets.

In some embodiments, heaters located above the droplets may obscure thedroplets thus interfering with real-time optical measurements. In suchcases, the droplets can be transported out from underneath the heatersto a location which is preferred for optical detection (i.e. a detectionspot). Droplets may be periodically transported out from underneath theheaters to a detection spot on the droplet microactuator detectionpurposes, e.g. detection by fluorescence quantitation. Droplets may berouted into proximity with a sensor while cycling them from onetemperature zone to another.

8.8.7 Droplet Microactuator Fabrication

Droplet microactuators can be made using standard microfabricationtechniques commonly used to create conductive interconnect structures onmicrodroplet microactuators and/or using printed-circuit board (PCB)manufacturing technology. Suitable PCB techniques include thosedescribed in U.S. patent application Ser. No. 11/343,284, entitled“Apparatuses and Methods for Manipulating Droplets on a Printed CircuitBoard,” filed on Jan. 30, 2006, the entire disclosure of which isincorporated herein by reference. These techniques permit the dropletmicroactuator to be manufactured in bulk at very low cost. Low costmanufacture enables economical production of droplet microactuators,even for use as one-use disposables. Thus, the invention provides amethod in which droplet microactuators are supplied to users ascomponents of disposable cartridges for use in systems of the invention.

Designs can also be implemented on glass or silicon using conventionalmicrolithography techniques with the capability of producing muchsmaller features than are typical in a PCB process. Even, for example,for a 1,572,864-reservoir droplet microactuator with 70 μm reservoirspacing and 3 fL reservoir volume, the minimum required lithographicfeature size is ˜0.5 μm which is well within the capabilities ofconventional microlithographic techniques currently used in thesemiconductor industry.

8.9 Systems

Fluid loading may be accomplished using droplet microactuator systems,such as illustrated in FIG. 18. Steps of a fluid loading protocol may beconducted using a droplet control system 1801. A set of computerexecutable instructions may be written which can be loaded into acontroller for execution of a loading protocol. Integrated systemsincluding the droplet control system 1801 and the protocol executionsystem 1802 may also be used. The droplet control system 1801 permits auser to control droplet microactuator system functions, such as dropletoperations and sensor operations for fluid loading protocols. Theprotocol execution system 1802 permits a user to execute softwareroutines that control droplet microactuator system functions, such asdroplet operations and fluid loading operations. The invention alsoprovides a method or computer useable instructions for conducting fluidloading processes or protocols. The programmable flexibility of theplatform permits assays to be rapidly optimized and allows conditionalexecution steps to be implemented. For example, calibrations,confirmatory tests, or additional controls can be executed if triggeredby a particular test result. In some embodiments, the system canintegrate sample preparation steps. Automation of the system andon-droplet microactuator operations enhance portability and enableassays to be performed more quickly and by personnel with minimaltraining, thereby reducing human error.

Referring further to FIG. 18, at a high level, each of the systems ofthe invention typically includes a processor or controller 1803, adroplet microactuator 1804, a sensor or detector 1805, input device(s)1806, output device(s) 1807, and software. U.S. Patent Application No.60/806,412, entitled “Systems and Methods for Droplet MicroactuatorOperations,” filed on Jun. 30, 2006, the entire disclosure of which isincorporated herein by reference, describes droplet microactuatorsystems which may be employed in conjunction with the dropletmicroactuator aspects of the invention. The droplet control systemincludes droplet control software run on a computer 1808 and programmedto display a droplet control interface for controlling dropletmicroactuator system functions. The protocol execution system includesprotocol execution software programmed to facilitate execution of a setof computer executable or computer useable instructions for controllingdroplet microactuator system functions to conduct fluid loading.

8.9.1 Controller

The system of the invention may include a controller 1803. Thecontroller serves to provide processing capabilities, such as storing,interpreting, and or executing software instructions. The controllermay, for example, be comprised of a digital signal processor (DSP) withmemory, a microcontroller or an application specific integrated circuit(ASIC). An example of a suitable DSP processor is the Analog DevicesBlackfin DSP processor.

The controller is electronically coupled to various hardware componentsof the invention, such as the droplet microactuator, any sensors, andany input and/or output devices. The controller may be configured andprogrammed to control data and/or power aspects of these devices. Forexample, with respect to the droplet microactuator, the controllercontrols droplet manipulation by activating/deactivating electrodes.This aspect of the invention is discussed further in Section 8.8.

The controller may further be electronically coupled to a separatecomputer system including a processor, input and output devices, datastorage medium, and other components. This arrangement is particularlyuseful in the droplet control system, in which the computer system isprogrammed to operate a droplet control user interface. In thisarrangement, the processor of the computer system may accept input viathe user interface and transmit instructions to the controller, e.g., toactivate/deactivate electrodes, to read electrodes, memory, and/orsensors, and the like.

In the protocol execution system, software for controlling the systemmay be loaded directly into and executed by the controller to cause thecontroller to control the droplet microactuator system functions. Inthis embodiment, the system can run autonomously, e.g., as a portable orhandheld system.

8.9.2 Droplet Microactuator

The system may include a droplet microactuator 1804, as describedfurther in Section 8.8. The droplet microactuator is electronicallycoupled to the processor such that the processor can control variousoperations of the droplet microactuator, such as droplet manipulationoperations.

8.9.3 Sensor

Various embodiments of the invention make use of sensors or detectors1805. Sensors may include sensors which are coupled to the dropletmicroactuator for the purpose of measuring parameters of interest on thedroplet microactuator such as the fluorescent or luminescent intensityat a location on the droplet microactuator where a reaction product maybe located. Sensors may also include sensors which monitor the status ofthe system such as droplet microactuator insertion sensors, lid latchsensors, ambient temperature sensors and the like. Output from eachsensor may be mapped to a specific memory location, and the processormust only query the mapped location to obtain a reading from the sensor.The sensor is mounted relative to the droplet microactuator and/orelectronically coupled to the droplet microactuator such that the sensorcan detect signals, such as electrical or light signals, from thedroplet microactuator. Sensors are discussed in more detail elsewhere inthis specification, e.g., see Section 8.11.

8.9.4 Input and Output Device(s)

Systems of the invention also include various input devices 1806 andoutput devices 1807. In certain embodiments, such as the protocolexecution system, certain input and output devices may be controlledusing a human-machine interface (HMI) controller.

8.9.5 Software

Each of the systems of the invention includes software. The softwareprovided on a storage medium is one aspect of the invention. Examples ofsuitable storage mediums include magnetic storage, optical storage,phase-change memory, holographic storage, molecular memory storage,battery or capacitor-backed SRAM and flash memory storage. The softwaremay be loaded in memory and/or in a processor. A system in whichsoftware of the invention is present in memory and/or a processor and/ora storage medium is also an aspect of the invention.

The software of the invention may be written in any of a variety ofprogramming languages, such as Visual C, Java and/or Python. The systemmay include an interpreter for translating droplet manipulation andother instructions from the high-level language into an intermediatelanguage for execution by the processor. Alternatively, software writtenaccording to the invention may be compiled into machine language using acompiler. The software interpreter and compiler for the language of theinvention are themselves novel aspects of the invention. As such, allforms of data storage, memory, and processors containing the interpreterand/or compiler are aspects of the invention.

The system can be programmed to execute a wide variety of protocolsinvolving any number of droplet manipulations. Multiple droplets can beindependently and simultaneously manipulated on a single dropletmicroactuator. The capacity to independently manipulate multipledroplets in parallel enables execution of complex protocols as a seriesof basic microfluidic instructions. Systems are scalable and may controltens, hundreds, thousands or more parallel droplet manipulations perdroplet microactuator. For example, at any one moment, up to a maximumof every control electrode on the droplet microactuator may be engagedin a droplet operation.

The system can be programmed to enable users to input instructions forthe execution of protocols. Existing protocols may be monitored andadjusted according to user requirements. Complex protocols can beimplemented in which the outcome of one or more steps determines theselection of one or more subsequent steps. For example, a droplet inwhich a certain measured result is positive may be transported forfurther processing, while a droplet in which a result is negative may bediscarded, or vice versa.

8.9.6 Portability

Referring to FIGS. 19A and 19B, in some embodiments, the analyzer isprovided as a portable device, such as a hand-held device 1900. FIG. 19Ashows the exterior of handheld device 1900 and FIG. 19B shows a slot1902 for insertion of a droplet microactuator (not shown), an opticalsensor 1904 for sensing optical signals from the droplet microactuator,and a lid latch 1906, which may be coupled to the system to indicatewhether the lid is open or closed. It is envisioned that the portableanalyzer may also be a tabletop device. The portability of the dropletmicroactuator systems of the invention facilitates point of care orpoint of sample collection use in a wide variety of settings in clinics,operating rooms, emergency rooms, small laboratories, and in the field(emergency response teams, accidents, disasters, battlefield,bioterrorism sites etc.) for rapid diagnostics that can lead to quickturn around times in critical situations.

8.10 User Interface

The droplet control system includes droplet control software programmedto display a droplet control interface for controlling dropletoperations on the droplet microactuator, controlling the sensor, whenpresent, and controlling other hardware associated with the dropletcontrol system. The system may also include software to facilitatecreation of a set of software or computer useable instructions forcontrolling droplet microactuator system functions, such as dropletoperations and/or sensor operations.

As illustrated in FIG. 20, the system may include a user interface 2000.The user interface is described further in related U.S. PatentApplication No. 60/806,412, entitled “Systems and Methods for DropletMicroactuator Operations,” filed on Jun. 30, 2006, the entire disclosureof which is incorporated herein by reference. The user interface maydisplay a map 2001, preferably an interactive map, of a dropletmicroactuator. The map may be used to interact directly with the dropletmicroactuator to manipulate droplets on the droplet microactuator toconduct a fluid loading protocol according to the invention. The map maybe used in a virtual mode to manipulate virtual droplets 2011 in aprogramming mode to develop and record subroutines for controllingdroplet microactuator functions and related hardware.

8.10.1 Droplet Control System and User Interface

The droplet control system includes droplet control software. Thedroplet control software is programmed to display a droplet controlinterface for controlling droplet operations on the dropletmicroactuator, controlling the sensor, when present, and controllingother hardware associated with the droplet microactuator system. Thedroplet control software permits a user to manipulate droplets on adroplet microactuator via a software driven user interface. As describedabove, an example of such an interface is illustrated in FIG. 20. Amongother things, the user interface may permit a user to view informationabout a droplet microactuator. The user interface may also facilitateinput by the user which controls functions of the droplet microactuatorand associated devices, such as associated sensors.

With respect to controlling droplet operations on a dropletmicroactuator, the software is programmed and the system is configuredto, among other things, drive control and reference electrodes on thedroplet microactuator to conduct the droplet operations. Dropletoperations, which are discussed further in Section 8.8 above, areeffected by applying a voltage to selected electrodes. The software andsystem may be configured to permit software loaded in the processor tocontrol activation of the selected electrodes by controlling theoperation of relays associated with the electrodes.

As shown in FIG. 20, the user interface 2000, which is displayed on anoutput device, may be programmed to display a graphical illustration ormap 2001 of a droplet microactuator design. The map 2001 may be based ona matrix or other configuration that defines the position of each of thecontrol electrodes and/or reservoirs. Components of the map may bedifferentiated by appearance, e.g., by shape, color, brightness,symbols, icons, etc. For example, in the map displayed in FIG. 20,unactivated droplet manipulation electrodes 2002 can be shown in a firstcolor (such as gray), activated droplet manipulation electrodes andreservoirs 2003 can be shown in a second color (such as red), andunactivated reservoirs 2004 can be shown in a third color (such asblue).

In a simple embodiment, the matrix is defined in a control file whichidentifies a row and column for each electrode and/or reservoir. When acontrol file is loaded, the system reads in the matrix definitions anddisplays the corresponding map of the matrix on the user interface.

The interface may display information about components of the map, whichmay also be stored in the control file. In one embodiment, the systemdisplays information about a component when it is moused over, selected,or otherwise electronically identified by a user. Information displayedmay, for example, include some or all of the following information:

-   -   component type, e.g., droplet manipulation electrode, reagent        reservoir, sample reservoir, etc.;    -   electrical connectivity information, e.g., electrode        enumeration, grounds, pinout number etc.;    -   adjacency relationships, e.g., in a polygonal electrode        arrangement;    -   representative geometry, for rendering the map in the user        interface;    -   design notes and/or other comments;    -   part numbers;    -   column and/or row position.

The system may also record the history of the activation of eachelectrode, so that the user may track the number of times an electrodehas been activated. History information may, for example, be displayedby mousing over or selecting an electrode. The system may be programmedto accept input from a user instructing history information to bedisplayed simultaneously for all electrodes.

To facilitate user interaction, a moused over or selected electrode 2002or other component may also cause the electrode or other component to behighlighted on the droplet microactuator map. This capability permits auser who is directly controlling droplet microactuator operations toreview information about each potential step by mousing over the dropletmicroactuator component prior to actually selecting and activating thedroplet microactuator component. The system may be programmed tohighlight a moused over component and a selected component differentlyso that a user may differentiate between the two.

The system may include a means 2007 for permitting a user to select themode of operation, e.g., select between a virtual or programming mode inwhich a program can be written for controlling a droplet microactuator,and an operation mode in which droplets are controlled directly on adroplet microactuator.

The system may include a means 2012 for permitting a user to select adroplet microactuator design for display. Alternatively, dataidentifying the droplet microactuator design may be included as acomponent of the droplet microactuator assembly or cartridge accessibleby the system upon coupling of the droplet microactuator assembly orcartridge to the system.

It should be noted that in some designs, more than one electrode may becoupled to the same electrical output. Such designs can be used tosimplify the design of the droplet microactuator. In such designs,selecting or mousing over one electrode from a common set may result inselection, highlighting and activation of all electrodes in the set.

Thus, in one embodiment, the system is programmed so that when a userselects an unactivated electrode 2002 on a microactuator map 2001, thesystem activates the electrode. For example, the system may beprogrammed and configured so that clicking on a representation of anelectrode on the map causes a voltage to be applied to a correspondingactual electrode on the droplet microactuator, thereby activating theselected electrode. In this way, a user can directly manipulate dropletson the droplet microactuator using the interface.

The droplet control system may permit a user to transport a droplet bysequentially clicking on a series of adjacent electrodes. Similarly, thesystem may permit a user to transport a droplet by selecting a virtualon-screen droplet 2011 and dragging the droplet to a virtual electrodeat a desired location on the droplet microactuator map. Moreover, thesystem may permit a user to transport a droplet by selecting a virtualon-screen droplet 2011, then clicking a virtual electrode at a desiredlocation on the droplet microactuator map. Other droplet microactuatorcomponents may be similarly controlled via a user interface.

The system may be programmed to display a representation of theelectrical control lines 2005 electronically coupled to the dropletmicroactuator components, so that when a user mouses over and/or selectsa component, the system highlights the electrical signal that isactivated as a result of being mapped to the component.

The droplet microactuator may be visually monitored, e.g., using amicroscope and video capture device. The user interface may beprogrammed to display a real-time image of the droplet microactuatorfrom the video capture device. Further, the droplet microactuator mapmay be superimposed over the real-time droplet microactuator image sothat a user can visualize droplet operations on the dropletmicroactuator as he or she interacts with the droplet microactuator viathe user interface.

Similarly, the system may be programmed to display virtual droplets 2011on the droplet microactuator map which illustrate actual behavior ofdroplets on a droplet microactuator which is being controlled by thesystem, and/or the system may be programmed to display virtual droplets2011 on the droplet microactuator map which illustrate predictedbehavior of droplets on a droplet microactuator, even though a dropletmicroactuator is not being directly controlled by the system.

The system may also be programmed to effect an “inverse output” 2006operation. In typical operation, the droplets are constantly connectedto a ground voltage/ground line. In the “inverse output” operation, thesignals are inverted so that the droplet is at a high voltage and theelectrodes are activated by setting them to ground potential. In otherwords, the “inverse output” operation switches the polarity of thesignals.

The system may also facilitate creation of a set of software or computeruseable instructions for controlling droplet operations on the dropletmicroactuator and controlling other functions of a droplet microactuatorand related hardware. The software instructions may, for example,include instructions for executing a protocol for processing andanalyzing a sample and outputting results of the analysis. The systemmay facilitate writing programs for controlling droplet microactuatorfunctions and related components, such as sensor components withoutinteracting with an actual droplet microactuator.

The system may, for example, include means for permitting a user tocreate a program with a set of instructions for execution by the dropletmicroactuator. Examples of suitable instructions include:

-   -   “on” for identifying electrodes that are to be actuated;    -   “frequency” to set the rate at which the steps are executed,        e.g., the timing of electrode activation/deactivation;    -   “wait” to permit the instructions to pause for a predetermined        period;    -   “loop” to loop steps in the program;    -   “voltage” to set the voltage being applied to the outputs.

Instructions can be provided as a byte-coded language which includesinstructions needed to conduct droplet manipulations and control otheraspects of the system. The instructions prepared by the system can berecorded in the assembly language and assembled into byte codes. Thebyte codes can be loaded into a system of the invention, e.g., aprotocol execution system, for execution. The system may include asoftware interpreter for interpreting the language for execution, e.g.,in a protocol execution system.

In a preferred embodiment, the system displays a series of buttons oricons 2008 that can be selected to add, insert, update, modify or deleteinstructions from a subroutine. The buttons or icons may, asappropriate, be accompanied by fields 2009 for the entry of parametersassociated with the instructions. For example, by clicking the “add”button, a command can be added at the end of a subroutine. By clickingan “insert” button, a command can be inserted within a subroutine. Byclicking a “modify” button, a command present in a subroutine can bemodified. By clicking a “delete” button, a command can be deleted.Further, a display field 2010, which may be editable, may be includedfor viewing, entering and/or editing code.

The system may display a simulated execution of a subroutine on thedroplet microactuator map, which outputs to the user a visual display ofthe effects of the command series selected. In other words, in asimulated execution mode, the software executes the steps of asubroutine but does not send an electrical signal to the dropletmicroactuator. In a preferred simulation mode, simulated droplets aredisplayed on the screen to illustrate to the user the actual effect ofthe program. In this way, a user can readily troubleshoot a subroutinewithout requiring interaction with a droplet microactuator.

8.10.2 Protocol Execution System and User Interface

The invention provides a protocol execution system. The protocolexecution system includes protocol execution software programmed tofacilitate execution of a set of software instructions for loadingfluid, controlling droplet operations on the droplet microactuatorand/or other functions of a droplet microactuator and related hardware.The protocol execution system provides the ability to execute protocolson a free-standing system, typically a portable or handheld system.

The protocol execution system is configured to control the dropletmicroactuator and any associated components. Pre-programmed instructionsmay be loaded into the controller which controls the system and anyassociated components. The protocol execution system may include variouscomponents for permitting a user to provide input to and obtain outputfrom the processor. The human-machine interface may be facilitated usinga HMI board. The HMI board typically includes a controller and variouselectronic components, such as buses and ports for electronicallycoupling input and output devices with the processor.

8.11 Sensors

The droplet microactuators and systems include sensors for measuringdroplet properties, such as physical properties, chemical properties,and electrical properties. In some embodiments, the sensors will includea sensing element arranged to interact with a droplet and/or a signalfrom a droplet; a transducing element, which converts output from asensor into a measurable signal; a means for transmitting the signal tothe processor. The processor may convert the signal into an outputrecognizable to a user.

The sensor element may be a component of the droplet microactuator,e.g., mounted on a top or bottom plate, positioned in the interior spaceof a droplet microactuator between top and bottom plates, ormanufactured as an integral component of the droplet microactuator,e.g., an integral component of top or bottom plates. In otherembodiments, the sensor element may be exterior to the dropletmicroactuator but arranged within the system in a manner which permitsthe sensor to receive a signal from on the droplet microactuator, e.g.,from a droplet on a droplet microactuator. For example, a sensor elementfor sensing photons may be arranged to receive photons from a droplet ona droplet microactuator. Where the system has a top plate capable oftransmitting photons from a droplet, the sensor may be arranged inproximity to the top plate for sensing the photons. Where the system hasa top plate not capable of transmitting photons from a droplet, the topplate may be provided with a window capable of transmitting photons, andthe sensor may be arranged in proximity to the window for sensing thephotons.

Illustrative examples of sensor configurations are provided in FIGS.21A-21D wherein the sensors may be provided in association with a bottomplate 2102, a top plate 2104, and electrodes 2106. FIG. 21A illustratesan optical sensor which may include use of a setup including an LED 2108and a photodiode 2110 for monitoring absorbance. FIG. 21B illustrates aluminometric sensor which may include use of a photomultiplier tube(PMT) 2112. FIG. 21C illustrates a potentiometric sensor 2114 whichtypically functions based on the measurement of a potential under nocurrent flow. FIG. 21D illustrates an amperometric sensor 2116 whichtypically functions by the production of a current when a potential isapplied between two electrodes.

It is important to keep in mind that, as noted elsewhere in thisdisclosure, the droplet microactuator may be supplied as a separatecomponent which can be coupled to a system by a user. Where sensors areexterior to the droplet microactuator, those sensors may in someembodiments be aligned such that upon coupling to the dropletmicroactuator system, the sensing elements are appropriately aligned todetect signals from the droplet microactuator, e.g., the photon sensoris aligned with the appropriate window and/or with the appropriatelocation on the droplet microactuator where the sensing step will beaccomplished in the course of a droplet protocol.

In various embodiments, the droplet microactuator and/or system may beconfigured with sensor components enabling the implementation of one ormore types of sensing. Examples of suitable sensing types includephysical sensing, electrochemical sensing, and optical sensing.

8.11.1 Physical Approaches

A droplet microactuator and/or system of the invention may include oneor more physical sensors arranged to sense a property of a droplet on adroplet microactuator. Examples of physical sensing include temperatureand droplet size (e.g., by thermally measuring the footprint of thedroplet).

8.11.2 Electrochemical Approaches

The droplet microactuator system of the invention makes use of a varietyof optical detection approaches. A droplet microactuator and/or systemof the invention may include one or more electrochemical sensorsarranged to sense a property of a droplet on a droplet microactuator.Examples of suitable electrochemical sensing types includepotentiometric sensors, amperometric sensors, voltametric sensors, andconductometric sensors. The various components of the sensors (e.g.,electrodes, counter electrodes, reference electrodes, etc.) may beprovided on the same or separate substrates, arranged to permit contactwith a droplet on the droplet microactuator. For example, in embodimentsin which the droplet microactuator includes two substantially parallelsubstrates, various components of the sensor assemblies may be comprisedon one or both of the substrates. In some embodiments, an electriccircuit may be used to amplify signals into a measurable voltage.Various aspects of these approaches are discussed in the ensuingsections.

8.11.2.1 Amperometry Sensor

The droplet microactuator device or system may include an amperometrysensor and an electrical source arranged to permit a droplet on thedroplet microactuator to be transported into contact with electricalsource and the sensor to permit detection of electric current flowingthrough the droplet.

8.11.2.2 Potentiometry Sensor

The droplet microactuator device or system may include a potentiometrymeasuring and reference electrode arranged to permit a droplet on thedroplet microactuator to be transported into contact with themeasurement and reference electrodes to permit measurement ofequilibrium electrode potential of a droplet.

8.11.3 Optical Approaches

The droplet microactuator system of the invention makes use of a varietyof optical detection approaches.

A droplet microactuator and/or system of the invention may include oneor more optical sensors arranged to sense a property of a droplet on adroplet microactuator. Examples of optical sensing include absorbance,chemiluminescence, and fluorescence. Optical sensors may in some casesbe accompanied with an appropriate light source, e.g., for excitingfluorescence or conducting absorbance measurements. These sensors may beprovided as components mounted on a droplet microactuator and/or asintegral parts of a droplet microactuator, e.g., using semiconductormanufacturing techniques.

Optical sensors may include various optics designed to direct opticalsignals, and may be coupled to various image processors for analyzingoptical images. For example, droplet size may be detected by processingan image of a droplet. Similarly, droplet size may be obtained bymeasuring a thermal footprint of the droplet. Electrical sensors mayalso be used to measure droplet size, e.g., by measuring impedance ofthe droplet footprint.

In some cases, surfaces of the droplet microactuator may be modified toenhance optical sensing. For example, electrodes with reflective surfacefinishes may be used to facilitate optical measurements of droplets. Theuse of reflective electrodes increases the path length for absorbancemeasurements and is also compatible with reflectance spectroscopy. Forauto-fluorescent substrates, such as PCB, coating a dropletmicroactuator surface with a non-fluorescent coatings can be used toprovide a non-fluorescent detection zone.

Various aspects of these approaches are discussed in the ensuingsections.

8.11.3.1 Photosensor

The droplet microactuator device or system may include an absorbancedetection components including a light source and a photosensor arrangedto permit a droplet on the droplet microactuator to be transported intoproximity with the light source and photosensor such that light orenergy passing through the droplet can be detected by the photosensor.

The droplet microactuator device or system may include chemiluminescencedetection components including a photosensor (such as a photodiode,avalanche photodiode, photomultiplier tube) or photon sensor (such as aphoton-counting photomultiplier tube) arranged to permit a droplet onthe droplet microactuator to be transported into proximity with thephotosensor or photon sensor such that photons emitted by chemicalspecies in the droplet can be detected by the photosensor or photonsensor.

8.11.3.2 Fluorescence Sensor

The droplet microactuator device or system may include fluorescencedetection components including a light excitation source withappropriate filters, if necessary, and a photosensor (such as aphotodiode, avalanche photodiode, photomultiplier tube) or a photonsensor (such as a photon-counting photomultiplier tube) with appropriatefilters and dichroic mirrors, if necessary, arranged to permit a dropleton the droplet microactuator to be transported into proximity with thelight excitation source and the photosensor or photon sensor such thatphotons emitted by fluorescent species in the droplet can be detected bythe photosensor or photon sensor.

8.11.3.3 Surface Plasmon Resonance

In another embodiment, surface plasmon resonance (SPR) sensing isemployed to detect interactions between an antibody and any targetanalyte. SPR sensing is useful to detect and quantify such interactions.Typically, one interactant in the interactant pair (i.e., antibody oranalyte) is immobilized on an SPR-active gold surface on a glasssubstrate. The interactant may be immobilized using a droplet-basedapproach wherein a droplet is transported into contact with the goldsurface to deposit the interactant thereon. A droplet including theother interactant may be transported into contact with the immobilizedinteractant, thereby permitting the other interactant to bind to theimmobilized interactant. When light (e.g., visible or near infrared) isdirected through the glass substrate and onto the gold surface at anglesand wavelengths near the surface plasmon resonance condition, theoptical reflectivity of the gold changes very sensitively with thepresence of biomolecules on the gold surface or in a thin coating on thegold. The optical response may be highly sensitive due to the fact thatit involves an efficient, collective excitation of conduction electronsnear the gold surface. The extent of binding between the solution-phaseinteractant and the immobilized interactant may be observed andquantified by monitoring this reflectivity change. The invention alsoincludes a droplet microactuator including a gold surface thereon, and apath or network of electrodes arranged to permit the execution ofdroplet manipulations sufficient to bring a droplet into contact withthe gold surface. Further, the invention includes a system includingsuch droplet microactuator and further including a light source capableof directing light onto the gold surface at angles and wavelengths nearthe surface plasmon resonance condition. Similarly, the inventionincludes a system including such a droplet microactuator and furtherincluding a means for detecting changes in reflectivity of the goldsurface. Moreover, the invention includes a droplet microactuator deviceand/or system having loaded thereon reagents sufficient to conduct someor all steps of an SPR protocol.

8.11.3.4 Raman Spectroscopy

In one embodiment, the droplet microactuator and/or system includesRaman spectroscopic detection capability. In general, this capabilityincludes a Raman signal-generating light source, a Raman signaldetection surface, and a Raman spectrophotometer.

The Raman signal generating light source may, for example, be amonochromatic light, e.g., a laser source with excitation in the visiblewavelength range. The light source is arranged to irradiate a Ramansignal detection surface on a droplet microactuator. The surface may,for example, be a surface of the droplet microactuator and/or a surfaceof a particle on a droplet microactuator. For example, the surface maybe the surface of a particle in a droplet on a droplet microactuator.The droplet microactuator may have the capability of conducting dropletoperations using a droplet including such particles in order to effectvarious protocols which employ Raman signal detection methods.

The Raman signal detection surface may include any surface appropriatefor Raman scattering. Examples include gold or silver surface. Thesurface may be roughened. The droplet microactuator may in some casesinclude multiple metallic surfaces (e.g., surfaces of the dropletmicroactuator, beads, particles, nanoparticles, etc.), includingsurfaces labeled with a different Raman reporter molecules. Antibodiesor analytes bound to the surface may be identified by the characteristicRaman spectra of the Raman reporter molecules. The Raman detectionsurface may, for example, be an electrode, a coating on the electrode,or a layer on any chip surface. In operation, a droplet is positionedusing droplet operations on the Raman detection surface, and isirradiated with a laser beam. Scattered light from the irradiatedsurface is collected with a spectrometer. In another embodiment, theRaman detection surface is a particle in a droplet on a dropletmicroactuator. The particle may, for example, be a nanoparticle, such asa silver or gold nanoparticle. For example, silver nanoparticles can beprepared as monodispersed colloidal suspensions, which can bemanipulated on a droplet microactuator using droplet operations. In someembodiments, the particles may be aggregated into clusters usingaggregation additives, such as inorganic salts such as sodium chlorideor sodium nitrate, acids such as nitric or hydrochloric or organicamines such as poly-L-lysine. These aggregation additives can becombined with a droplet including the sample and the particles usingdroplet operations, e.g., using droplet operations to combine a dropletincluding the aggregation additive with a droplet including theparticles and sample. Surfaces of the droplet microactuator associatedwith the Raman spectroscopic region are selected to minimize thebackground fluorescence signal.

A Raman spectrophotometer is arranged to detect Raman scattered lightemitted from the sample droplet. The Raman spectrophotometer may beintegral with the droplet microactuator arranged exterior to the dropletmicroactuator in a manner which permits it to detect Raman scatteredlight emitted from the sample droplet on the droplet microactuator.

In operation, a droplet microactuator is provided having a Ramandetection surface thereon. An analyte is brought into association withthe Raman detection surface using droplet operations. The surface isirradiated with a Raman signal generating light source. Raman scatteredlight signals are detected correlated with expected signals in order todetermine the identity and/or quantity of an analyte.

In another embodiment, surface-enhanced Raman scattering (SERS) isemployed to detect interactions between an antibody and any targetanalyte. In general, this method involves monitoring an analyte mediatedbinding event in a sample droplet which includes the analyte, a specificbinding member, a Raman-active label, and is in contact with a surface,such as a bead or a surface of the droplet microactuator, and which iscapable of inducing a surface-enhanced Raman light scattering. Thesample droplet is illuminated with a radiation sufficient to cause theRaman-active label in the test mixture to emit a detectable Ramanspectrum. The differences in the detected surface-enhanced Ramanscattering spectra are dependent upon the quantity of the analytepresent in the test mixture. The presence and/or quantity of the analytein the sample droplet may be determined by monitoring the Ramanscattering spectra. The invention includes a droplet microactuatordevice and/or system having loaded thereon reagents sufficient toconduct some or all steps of an SERS protocol.

In a related embodiment, the invention provides a method for determiningthe presence or quantity of an analyte in a sample droplet by monitoringan analyte-mediated ligand binding event on a droplet microactuator. Themethod generally includes reacting the analyte with an antibody coupledto a Raman active label. The reaction is conducted using droplets on adroplet microactuator and is effected under conditions permittingspecific binding of the antibody to the analyte, if present, to yield afirst complex in the sample droplet. Sequentially or simultaneously thefirst complex is contacted with a surface capable of inducing asurface-enhanced Raman light scattering and having attached thereto anantibody specific for the analyte to form a second complex. The secondcomplex is illuminated with a radiation sufficient to cause theRaman-active labels in the complex to produce a detectable Ramanspectrum. Differences in the surface-enhanced Raman scattering spectraare indicative of the presence and/or quantity of the analyte present inthe test mixture.

A variety of surfaces may be appropriate for the droplet-based SERSprotocols of the invention. Examples include roughened metal electrodes,aggregated, films, metal islands of different morphology, semicontinuousfilms near the percolation threshold, and vacuum-evaporatednanostructured metal films. Accordingly, the invention includes adroplet microactuator including an SERS substrate. The dropletmicroactuator is suitably arranged such that a droplet may betransported along a path or network of electrodes into contact with theSERS substrate.

In DNA detection methods of the invention, a Raman label may be used. Alabel may be a non-sequence specific intercalator or a specific labelcovalently attached to a unique probe sequence. Negatively chargedlabels may require the use of a charge neutralizing agent, such asspermine, to facilitate association of the label with a negativelycharged surface, such as silver nanobeads with a citrate surface layer.Aggregating agents may also be used in order to improve signal. Sperminemay also serve as an aggregating agent.

8.11.3.5 Multisensor Capabilities

Preferred sensors are sensors for detecting absorbance, fluorescence,chemiluminescence, as well as potentiometric, amperometric, andconductometric sensors. The droplet microactuator device and/or systemof the invention includes one or more of these detection capabilities.In one embodiment, a droplet microactuator includes components forfacilitating 2 or more of these detection methods on a single dropletmicroactuator. In another embodiment, the droplet microactuator includesone detection module, but the system is programmed to conduct more thanone test using the module. In this embodiment, processed sample dropletsrequiring testing are sequentially moved into position for testing.Thus, multiple samples may be multiplexed over a detection spot where asingle sensor is used.

8.11.4 Sensor Electronics

The detection capabilities may be provided as one or more components ofa sensor board. The sensor board may include one or more sensors. Thesensor board may include additional electronic circuitry such asamplifiers, A/D converters, read-out circuits and the like forconditioning or amplifying the signal received from a droplet. Thesensor board may include control elements or other off-dropletmicroactuator components of the detection protocol, such as control ofmotors for moving components of the system.

In one embodiment, the sensor board includes a servo motor controllerfor controlling a servo motor that moves a magnetic field source intoand out of proximity with the droplet operation surface, therebyapplying/removing the magnetic field to/from the droplet microactuator.This embodiment is useful for manipulating magnetically responsivematerials. The sensor board may also include power supply elements andcommunication elements, including without limitation, elements requiredto electronically couple the sensor components or control components ofthe board to the processor.

The optical detection location may include specialized coatings,electrode designs, or other features that facilitate optical detection.For example, the detection spot may include a specialized pad and/orcoating that facilitates its operation as a background surface foroptical measurement.

In certain embodiments, such as nucleic acid amplification applications,the preferred optical detection method is fluorescence quantitation. Insuch embodiments, the detection spot may be selected to shieldbackground fluorescence present in the microactuator substrate orcoatings disposed on the microactuator substrate. For example, in oneembodiment, the microactuator is comprised of a printed circuit boardsubstrate and the detection spot is comprised of a gold pad whichshields the background fluorescence of the substrate from the sensorthereby facilitating fluorescent measurement of a droplet positioned onthe pad. The pad may be formed in a metal layer disposed directly on thesubstrate or disposed on an intervening layer disposed on the substrate.

Preferably, the metal layer in which the pad is formed should bedisposed on top of any layers exhibiting significant backgroundfluorescence. In one embodiment the pad is disposed directly on aprinted circuit board substrate being formed in the same metal layer asthe electrodes for controlling the droplet. In this embodiment, thedielectric material (which may also exhibit background fluorescence) maybe disposed above the metal layer and is selectively removed from thedetection pads, but not the control electrodes.

Thus, a low background fluorescence detection spot may be achievedthrough a combination of selective removal of fluorescent material abovethe detection pad and optical shielding of fluorescent material locatedbelow the pad. The pad is preferably designed to minimize itsinterference with other droplet microactuator functions. In theembodiment described above, the pad may be formed in the same metallayer as the control electrode but is separate and electrically distinctfrom the control electrodes. The pad therefore

8.11.5 Detection Approaches

The invention provides a variety of approaches for sensing/detectingsignals or attributes of droplets. Many of these approaches aredescribed elsewhere in this specification. This section describesadditional approaches that may be useful in various settings.

An advantage of the droplet microactuator approach of the inventionincludes the ability to decouple reaction steps in a particular assay.Many biochemical assays use common end reactions to generate a color,light or other detectable quantity. Droplet-based protocols of theinvention can be used to combine the assay droplet with a dropletcontaining the end reaction reagents at the point of detection.Decoupling of the assay steps permits each to be separately optimizedand separation of the steps in time provides greater flexibility whenone of the reaction steps is rate limiting. For examplechemiluminescence assays typically have better results at a basic pH.For an assay which is optimal at an acidic pH assay reaction can becompleted first at the acidic pH, and the light generation aspect of thereaction can be performed at a basic pH.

The droplet microactuators of the invention are useful in the study ofrate kinetic reactions. Sample droplets can be periodically dispensedfrom a reservoir in which a reaction is occurring. The droplets can thenbe individually analyzed to determine the time course of the reaction.The droplets can be analyzed in real-time or mixed with another reagentfor later analysis. Electrowetting may also be used to rapidly mixdroplets for the purpose of studying fast reaction kinetics.

Changes in viscosity of a droplet can be measured as a means forassessing the state of a chemical reaction inside the droplet. Forexample, a coagulant can be added to a droplet of blood followed bytransporting the droplet and monitoring of the ease of transport of thedroplet. Greater degrees of coagulation would make transport of thedroplet more difficult which can be detected as used as a measure of thedegree of coagulation.

Preferred sensors include optical sensors for sensing optical signals,such as absorbance, fluorescence, and chemiluminescence, andelectrochemical sensors for sensing electrochemical properties, such aspotentiometric properties, amperometric properties, conductometricproperties. Accordingly, the droplet microactuator system of theinvention includes components arranged to facilitate detection of one ormore of these properties. In one embodiment, 2 or more of theseproperties are detected on one or more droplets on a single dropletmicroactuator or otherwise accomplished using a single dropletmicroactuator system. In another embodiment, the droplet microactuatorincludes one sensor of a particular type, and the system is programmedto conduct more than one test using the sensor. In this embodiment,processed sample droplets requiring testing are sequentially moved intoposition for testing, i.e., moved into sufficient proximity to therequisite sensor to enable detection. Thus, multiple samples may bemultiplexed over a detection spot for detection by a single sensor.Multiple sensor types may be supplied on a single droplet microactuatorusing this approach.

The droplet microactuator system may in some embodiments be configuredto deposit a droplet or sample to a location that is exterior to thedroplet microactuator for detection. For example a droplet including (orpotentially including) an analyte can be deposited on a substrate forMALDI-TOF analysis.

Droplets can be cyclically transported past a common detection point inproximity to an appropriate sensor to allow multiple reactions to besimultaneously monitored. For example the droplet microactuator caninclude two or more “tracks” that connect high and low temperature zonesin a flow-through PCR reaction chamber. A single detector is placed atthe intersection of the tracks. Droplet traffic can be timed to causedroplets to sequentially pass over the detection spot.

Examples of assays suitable for execution in the droplet-based protocolsof the invention on the droplet microactuator of the invention includeoptical assays, such as absorbance assays, fluorescence assays,bioluminescence assays and chemiluminescence assays; and electrochemicalassays, such as potentiometric assays, amperometric assays, andconductometric assays. For example, various combinations of one or moreof the foregoing assay types can be used to identify and/or quantify oneor more analytes, such as proteins, enzymes, nucleic acids, metabolites,electrolytes, gasses (e.g., blood gases) and hematocrit. A system of theinvention may be programmed to conduct on a single droplet microactuatorvarious combinations of these assay types.

In one embodiment, a single droplet microactuator or system includesdetection capabilities for 2, 3, 4, 5, 6 or more different kinds ofassays. For example, the droplet microactuator device, system and/orother components of a droplet microactuator system may separately ortogether include one or more detection components, such as componentsfor amperometry, potentiometry, conductometry, absorbance,chemiluminescence, fluorescence, and/or temperature. Further, a dropletmicroactuator system may be programmed to execute assay protocols forconducting 2, 3, 4, 5, 6 or more different kinds of assays on the sameor multiple samples or sample types.

Within the droplet microactuator, the droplet manipulation componentsand the detection components may in some embodiments be decoupled byproviding them on separate substrates. Similarly, various detectioncomponents may be provided as part of a droplet microactuator device orsystem, but separate from the droplet microactuator. Thus, for example,a sensor may be provided on a cartridge to which the dropletmicroactuator is coupled. The coupling is arranged so that when thedroplet microactuator is coupled to the cartridge, suitable componentsare aligned to permit detection. Thus, for example, a photon sensor maybe aligned with a window or other transparent substrate so that when thedroplet microactuator is properly mounted on the cartridge, photonsemitted from a droplet on the droplet microactuator may pass through thewindow or substrate for detection by the photon sensor. Similarly, wherea light source is necessary to cause fluorescence of a molecule in adroplet on the droplet microactuator, the light source may be mounted tothe cartridge or other component of the droplet microactuator device orsystem and aligned so that the light source can reach the droplet toproduce the desired fluorescence.

In one embodiment, the droplet microactuator includes electrodes forperforming electrochemistry. Electrodes can be patterned onto theelectrowetting substrate to permit electrochemical measurement ofdroplets in contact with the electrodes. In a two-substrate dropletmicroactuator, the electrodes for performing electrochemistry can beformed either or both substrates. In some embodiments, transportelectrodes and electrochemical measurement electrodes are provided ondifferent substrates. The electrodes may include membranes forfabricating ion-selective analyses.

8.12 Other Methods

The invention includes a method in which components of a bench-topsystem are offered to or provided to a customer in exchange forconsideration. In one embodiment, the components offered to or providedto the customer do not include the PC. The software of the invention maybe provided to the user on a storage medium or made available fordownload via a network, such as the Internet. The user may obtain othercomponents of the system, couple the components to a PC, load thesoftware on a PC, and thereby assemble system of the invention.

The invention includes a method in which a bench-top system is used togenerate code for executing a protocol. Code is uploaded into a separatesystem, such as a portable or handheld system, which is offered to orprovided to a customer in exchange for consideration. The user may usethe system for executing the protocol.

The invention also includes a method in which programming and/or systemcontrol is effectuated remotely via a network, such as a telephonesystem or the internet. Thus, for example, a system may be sold to auser, a programmer may connect to the system via a user interfacedisplayed via the Internet to control the system, create programs usingthe system, load programs on the system, and/or repair programs on thesystem. As another example, the invention includes a process whereby aremote user accesses a droplet microactuator via a network and performsone or more droplet manipulations on the system.

8.13 Kits

A further aspect of the invention is a kit including reagents, samplecollection devices, and/or a droplet microactuator or cartridge forconducting the methods of the invention.

8.14 Concluding Remarks

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of theinvention. Other embodiments having different structures and operationsdo not depart from the scope of the present invention.

As will be appreciated by one of skill in the art, the present inventionmay be embodied as a method, system, or computer program product.Accordingly, the present invention may take the form of an entirelyhardware embodiment, an entirely software embodiment (includingfirmware, resident software, micro-code, etc.) or an embodimentcombining software and hardware aspects that may all generally bereferred to herein as a “circuit,” “module” or “system.” Furthermore,the present invention may take the form of a computer program product ona computer-usable storage medium having computer-usable program codeembodied in the medium.

Any suitable computer useable medium may be utilized. Thecomputer-usable or computer-readable medium may be, for example but notlimited to, an electronic, magnetic, optical, electromagnetic, infrared,or semiconductor system, apparatus, device, or propagation medium. Morespecific examples (a non-exhaustive list) of the computer-readablemedium would include some or all of the following: an electricalconnection having one or more wires, a portable computer diskette, ahard disk, a random access memory (RAM), a read-only memory (ROM), anerasable programmable read-only memory (EPROM or Flash memory), anoptical fiber, a portable compact disc read-only memory (CD-ROM), anoptical storage device, a transmission medium such as those supportingthe Internet or an intranet, or a magnetic storage device. Note that thecomputer-usable or computer-readable medium may even be paper or anothersuitable medium upon which the program is printed, as the program can beelectronically captured, via, for instance, optical scanning of thepaper or other medium, then compiled, interpreted, or otherwiseprocessed in a suitable manner, if necessary, and then stored in acomputer memory. In the context of this document, a computer-usable orcomputer-readable medium may be any medium that can contain, store,communicate, propagate, or transport the program for use by or inconnection with the instruction execution system, apparatus, or device.

Computer program code for carrying out operations of the presentinvention may be written in an object oriented programming language suchas Java, Smalltalk, C++ or the like. However, the computer program codefor carrying out operations of the present invention may also be writtenin conventional procedural programming languages, such as the “C”programming language or similar programming languages. The program codemay execute entirely on the user's computer, partly on the user'scomputer, as a stand-alone software package, partly on the user'scomputer and partly on a remote computer or entirely on the remotecomputer or server. In the latter scenario, the remote computer may beconnected to the user's computer through a local area network (LAN) or awide area network (WAN), or the connection may be made to an externalcomputer (for example, through the Internet using an Internet ServiceProvider).

The present invention is described with reference to flowchartillustrations and/or block diagrams of methods, apparatus (systems) andcomputer program products according to embodiments of the invention. Itwill be understood that each block of the flowchart illustrations and/orblock diagrams, and combinations of blocks in the flowchartillustrations and/or block diagrams, can be implemented by computerprogram instructions. These computer program instructions may beprovided to a processor of a general purpose computer, special purposecomputer, or other programmable data processing apparatus to produce amachine, such that the instructions, which execute via the processor ofthe computer or other programmable data processing apparatus, createmeans for implementing the functions/acts specified in the flowchartand/or block diagram block or blocks.

These computer program instructions may also be stored in acomputer-readable memory that can direct a computer or otherprogrammable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory produce an article of manufacture including instruction meanswhich implement the function/act specified in the flowchart and/or blockdiagram block or blocks.

The computer program instructions may also be loaded onto a computer orother programmable data processing apparatus to cause a series ofoperational steps to be performed on the computer or other programmableapparatus to produce a computer implemented process such that theinstructions which execute on the computer or other programmableapparatus provide steps for implementing the functions/acts specified inthe flowchart and/or block diagram block or blocks.

This specification is divided into sections for the convenience of thereader only. Headings should not be construed as limiting of the scopeof the invention.

It will be understood that various details of the present invention maybe changed without departing from the scope of the present invention.Furthermore, the foregoing description is for the purpose ofillustration only, and not for the purpose of limitation, as the presentinvention is defined by the claims as set forth hereinafter.

We claim:
 1. A method of detecting a target analyte in a sample, themethod comprising: (a) executing electrowetting-mediated dropletoperations and thereby: (i) combining (A) one or more immunoassayreagent droplets comprising magnetically-responsive beads havingaffinity for the target analyte with (B) one or more sample dropletspotentially comprising the target analyte to yield (C) a first combineddroplet; (ii) beginning with the combined droplet, effecting adroplet-based washing protocol to wash the magnetically-responsive beadsto yield a first washed droplet comprising the washed magneticallyresponsive beads; and (iii) combining (A) the droplet comprising thewashed magnetically responsive beads with (B) a droplet comprising areporter molecule having affinity for target analyte captured on themagnetically-responsive beads to yield (C) a second combined droplet;(iv) beginning with the second combined droplet, effecting adroplet-based washing protocol to wash the magnetically-responsive beadsto yield a second washed droplet comprising the washed magneticallyresponsive beads; (b) detecting a signal from the second washed dropletwhich corresponds to the presence, absence and/or quantity of theanalyte in the sample.
 2. The method of claim 1 wherein the detectingstep comprises detecting a signal from the second washed droplet.
 3. Themethod of claim 1 wherein the signal measured corresponds to unoccupiedbinding sites on the one or more magnetically responsive beads.
 4. Themethod of claim 1 wherein the signal measured corresponds to occupiedbinding sites on the one or more magnetically responsive beads.
 5. Themethod of claim 1 wherein the reporter molecule comprises a labeledantibody.
 6. The method of claim 5 wherein the labeled antibodycomprises a radioactive isotope.
 7. The method of claim 1 wherein thereporter molecule comprises an antibody conjugated to an enzyme capableof catalyzing a detectable reaction.
 8. The method of claim 7 whereinthe detectable reaction result in a colorimetric, fluorescent and/orluminescent property or signal.
 9. The method of claim 1 wherein the oneor more sample droplets comprise a substance selected from the groupconsisting of whole blood, lymphatic fluids, serum, plasma, sweat, tear,saliva, sputum, cerebrospinal fluids, amniotic fluids, seminal fluids,vaginal excretions, serous fluids, synovial fluids, pericardial fluids,peritoneal fluids, pleural fluids, transudates, exudates, cystic fluids,bile, urine, gastric fluids, intestinal fluids, fecal samples, fluidizedtissues, fluidized organisms, biological swabs and biological washes.10. The method of claim 1 wherein the method further comprises providingthe sample droplet by conducting electrowetting-mediated dispensing ofthe sample droplet on the droplet actuator.
 11. The method of claim 1wherein the signal comprises an electrochemical property of a droplet ona droplet microactuator.
 12. The method of claim 11 wherein theelectrochemical property comprises an amperometric, potentiometricand/or conductometric property of a droplet on the dropletmicroactuator.
 13. The method of claim 1 wherein the signal comprises anoptical property of a droplet on a droplet microactuator.
 14. The methodof claim 13 wherein the optical property comprises absorbance,chemiluminescence and/or fluorescence.
 15. The method of claim 13wherein the optical property comprises Raman spectra.
 16. The method ofclaim 1 wherein the signal comprises a physical parameter of a dropleton a droplet microactuator.
 17. The method of claim 16 wherein thephysical parameter comprises temperature.
 18. The method of claim 16wherein the physical parameter comprises emission of radioactiveparticles.